M465:Counting

Starting the Isolation & Study of Culturable Bacteria from your habitat

Background

There are large numbers of both beneficial and non-beneficial bacteria in animal guts. Often their roles are not well understood. Since we are limiting our focus to bacteria, we will choose media that encourage growth of bacteria and/or discourage growth of fungi and other eukaryotic microorganisms. A wide variety of growth media and incubation conditions can be used to isolate bacteria from soil. In general, we can favor the growth of certain groups over others by altering the composition (e.g. pH, osmolarity) and/or nutrients available. Some bacteria will grow so fast on rich media (nutrient agar, TSA, etc.) that they will mask other slower growing genera. We will try not only enriched nutrient and dilute nutrient agar but also a variety of defined media that will favor slower growing genera.

The vast majority of bacteria (90-99%) will not grow on your plates at all. We will only find the ‘culturable’ bacteria that like the growth conditions you choose. The more types of media and growth conditions you use, the greater the variety and number of bacteria that you will find. Although most habitats contain a rich and unbelievably diverse community of microorganisms, we will focus part of our investigation on a few of the culturable bacteria that contribute to this unseen world. To get a sense of the true diversity of the bacterial community in your habitat, later on in the semester, we will isolate genomic DNA and ID bacteria by 16S rRNA gene sequencing.

Activity 1: Plate Count of Bacteria: Assessing Culturable Bacteria from Your Habitat

To perform a standard plate count of your sample bacteria you must first serially dilute the sample so that the colony numbers will be manageable and so that you are more likely to have a countable number of cells. If you do not remember how to perform a dilution series, check out this useful link dilutions.

Your extract will have been frozen at a 1:100 dilution (in 10% glycerol, 1 gram/100 ml). This could also be called a 1% (w/v)solution.
Gather or find the following materials:

1. 5 sterile culture tubes with caps
   
2. 1 sterile disposable plastic individually wrapped 1ml pipet,
   
3. 5 sterile dilute nutrient agar plates,
   
4. 5 sterile plastic disposable spreaders

Setting Up a Standard Plate Count:
1. Label 5 tubes 10^-3, 10^-4 etc. through 10^-7.

2. Label 5 destination plates of appropriate solid medium with dilution and identifying information. Because your goal is to obtain 30-300 well isolated colonies on a plate, generally only the 10^-4 through 10^-7 dilutions are plated. However, we are going to plate all of our dilutions.

3. Slightly dehydrate the medium on each plate by cleaning the laminar flow hood, turning on the fan, and placing the plates in the hood, positioning the covers so they are ajar for 10 minutes or until the medium surface shows no visible moisture.

4. Pipet 0.9 ml of sterile water into the 5 tubes labeled in step 1. (You may use the same sterile 1ml pipet for all of them.)

5. Using your P200 micropipet, transfer 100μL of the 1:100 dilution to the tube labeled 10^-3, mix well by vortexing.

6. Using a new tip, transfer 100 μL ml of the 10^-3 dilution to the tube labeled 10^-4. Mix well. Mixing 0.1ml of the 10^-3 dilution with 0.9ml of sterile water makes a 10^-4 dilution.

7. Continue to transfer 100μL aliquots (after mixing well) from each dilution to the next tube of water until you have carried the dilution to 10^-7.

8. Use your P200 micropipet and a new tip to transfer 100μL of the 10^-3 dilution and dispense it to the center of the dilute nutrient agar plate labeled 10^-3. Use a sterile plastic disposable spreader to gently push the dispensed sample two or three times clockwise around the dish, and then several times counterclockwise. Make sure all of the surface area of the plate has been inoculated. Don't press too hard as force will cause the microorganisms to collect at the edge of the spreader, resulting in uneven distribution.

9. Repeat step 8 to inoculate the rest of your prelabeled dilute nutrient agar plates with the 10^-4 through 10^-7 dilutions of bacteria.

10. Allow the moisture to be absorbed into the agar before inverting the plates and putting a labeled piece of your team color tape around the set. Incubate the set of standard plate count plates at 37C in a rack designated by your instructor.

11. Repeat steps 8 and 9 for all media you will be inoculating as part of this project.

The formulation of enrichment media supplies specific nutrients that encourage the growth of bacteria types that grow too slowly or not at all in media missing these nutrients. Selective media is selective because it contains one or more ingredient(s) that inhibits the growth of competitor microbes (such as cycloheximine, a drug that prevents fungal growth but does not affect bacteria negatively). Media can be both selective and enrichment. In some cases growth on enrichment or selection media will take up to 2 weeks. Thus, you will have to keep track of the progress of your isolation of different bacterial species as not all of your organisms will be ready for the same steps at the same time.

Next week you will count the number of colonies on these plates, observe the variability in colonies on each plate and, possibly, select one or more of these colonies for isolation and further study.

Calculating the number of bacteria per gram of soil
If you divide the number of colonies you find by the amount of diluent used times the dilution factor, you will obtain the number of cultivatable bacteria per gram of soil. Only plates that with between 30 and 300 colonies per plate give accurate calculations.

CFU = number counted on plate/(diluent plated*dilution of plate counted)

Activity 2: Streaking for Isolation

How did your first attempt at streaking for isolation turn out? Have you checked your plate that you streaked in microbiology boot camp? If you didn't get single well isolated colonies of two different bacteria on that plate, ask your instructor to watch you prepare your first streak plate today so they can offer suggestions for improving your technique.

Come to the lab and check on your cultures often over the next few days. Make observations about the number, size, color and shape of the various colonies that appear and draw the growth you observe in your lab notebook. This project is an investigative one that will require outside of lab time. Because bacteria have widely varying generation times, they will form colonies at different rates. It is your responsibility to check your cultures often and subculture or move them to your lab section's designated rack in the walk-in cold room to alt growth before the isolated colonies we seek become a mess of overgrown lawn growth on your plates and the isolation must be started all over.

These enrichment protocols vary in the length of time between steps. We can't make this become regular once a week work to fit our lab schedule. You will need to be highly organized and remember when you need to come to lab to do the next part of the isolation or characterization tests. Fortunately, much of what you will need to do outside of lab time is not time consuming. Usually, it will amount to taking a well-isolated colony and subculturing it onto new media (a few minutes of work); however, your lab instructor can't keep track of all the isolations in progress and remind you that it is time for the next step. The success of this project depends on your organizational skills and your commitment to time-sensitive attention to the task at hand.