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Syllabus

• Agar slant tubes
• Aseptic technique
• Assays
• Assays Illustrated
• Colony morphology
• Gram stain
• Isolating colonies
• Making serial dilutions
• Culture media
• Preparing spread plates
• Relationship to oxygen
• Viewing Gram stains
• Viewing living bacteria

• Characterization writeup
• Lab responsibilities
• Project proposal guidelines
• Using Bergey's

Supplemental pdfs

• Inoculating sticks
• Microscopy procedures

Isolating bacterial colonies from a spread plate

Obtaining isolates from a plate with mixed colonies is fairly straightforward most of the time, provided the colonies are well separated. Our project goals, however, require more than simply transferring a colony or two to a fresh agar plate. We want to obtain representative cultures from all of the common, cultivable species inhabiting our water source. Thus we need to systematically screen our plates for unique colony types, obtaining as many unique cultures as we can. Presumably, each unique colony type represents a species or a particular strain of a species. For the sake of simplicity we will use the term "species" to refer to any unique colony type or culture.

We will want to record information such as how common the species was among the plates, how was it influenced by temperature of incubation, etc. Because we need to distinguish unique species from the beginning, we need to document the characteristics of each colony that we "rescue" from our spread plates.

Screen your plates

You will have two plates for each dilution ranging from 10-1 to 10-4, one set incubated at 35˚C and one at room temperature (RT). You should mark the RT plates so that you will be able to document where a species came from (dilution and incubation temperature).

Make general observations on the results. On which plates did you get good colony separation? What were the estimated #colonies on each plate? A rough estimate is all we need. Did any particular species show aggressive growth?

Use the phenotypic characteristics described in the document "Morphology.pdf" to distinguish unique colony types. View from the top if you can and also from the bottom of the plate. Use a dissecting microscope with transilluminator to check for opacity and to compare colonies that look similar but are not quite identical.

Document everything that might be useful later, such as frequency with which the colony type appeared on the plates, whether or not you obtained it from a crowded or uncrowded plate, and/or effect of temperature on growth, if any. Record the dilution and incubation temperature.

As you identify unique colonies for sampling, begin selecting colonies to rescue. If you can, select colonies that are well separated from the rest. Mark the colony position on the bottom of the plate with an x or a circle. If there is space you might want to number it in case you want to go back to the original plate and locate the same colony later.

After surveying the plates and identifying all of the colony types that you can distinguish with the resources at your disposal, it is time to begin collecting isolates.

Isolate your selected colonies

To obtain an isolate, flame-sterilize an inoculating loop or needle or use a sterile disposable toothpick or stick. Exercising aseptic technique, touch the colony so that some material adheres to the end of the instrument. Aseptically transfer the material to a fresh agar plate and streak it out using the dilution streak method.

Most colonies have a soft texture and can be sampled readily. Use a loop if you have plenty of clearance. Then you can use the same loop to do the dilution streaking. Use a needle if a loop is too big to enable you to sample from just that one colony.

INSERT IMAGE

Fig 1. Flaming a loop. Hold the loop horizontally in the top narrow blue part of the cone, then tilt up to a 45 degree angle and heat the loop and wire so that it glows red-orange. After flaming the loop, allow it to cool for about 10 sec before contacting a living culture. You may also cool the loop by touching it to a clean agar surface. http://www.biologyjunction.com/aseptic_techniques.htm

You may encounter a very dry colony or one with a hard surface that is difficult to penetrate. In that case you may be able to obtain material by poking through the colony with a needle and/or breaking it up with a sharp sterile instrument. Colonies of some species adhere so tenaciously to the agar surface that the only practical way to transfer material is to aseptically remove a plug of agar and transfer it to a new plate.

Streaking a plate

Even if a colony on a mixed plate is well separated from the others, it may have surrounded viable cells from other species. You may also inadvertently pick up material from a different colony type as you collect your isolate. To ensure that we obtain pure cultures we spread colony material onto a fresh agar plate using a dilution streaking method. Done properly, dilution streaking produces well separated colonies in at least one section of the agar, revealing the presence of contaminants.

Fig. 2. Examples of good and bad dilution streak technique. http://www.biologyaspoetry.com/terms/streak_plate_method.html

The objective of dilution streaking is to spread the colony material (the inoculum) over a large area of agar, spreading it thinner from section to section. Aseptically transfer some material from your instrument to the surface of a fresh agar plate, near one edge. If you have the material on a loop then you can continue to spread the sample using the same loop. Otherwise, flame and cool a loop to use for the spreading. Now follow the steps outlined below.

1. Resting the loop lightly on the surface, sweep it across the agar back and forth from left to right to spread the sample while gradually drawing the loop toward you. It is okay to overlap. The idea is to spread the sample over the surface, completing around 20-30 sweeps per section. The film of material you are spreading will become thinner as you draw the loop toward you.

***Don't forget to keep the lid over the agar while you are streaking out the sample***

2. When you have spread the sample about halfway to the center of the plate (~1/4 of the plate diameter), pick up the loop and flame it again. After cooling the loop, streak a new section of the plate.

a. Start by resting the loop on the surface as before, then sweep the loop back and forth, overlapping the previously inoculated section for the first two or three sweeps.

b. Now continue to spread the sample as you did the first time, but do not continue to overlap the previously inoculated section. This way, you are spreading a much smaller inoculum. You have, in fact, diluted the sample, thus the name "dilution streaking."

c. For three-way streaking, have your second section cover half of the remaining agar surface. Cover less if you plan a four-way or five-way streak plate. Figure 2 (above) shows a pattern for four-way dilution streaking

3. Repeat step 2 for one to three more sections, flaming and cooling the loop between sections.

4. Invert and incubate the plate and after the colonies have grown up inspect to see whether or not you have a pure culture. Figure 3 shows the sorts of results you might expect.

Fig.3. Dilution streak plates after 2 days incubation. (left, top arrow) A well separated colony of the species the investigator intended to isolate (4-way streak). (left, bottom arrow) Evidence that the culture is contaminated by at least one additional species. (right) Uniform appearance of colonies on a 5-way streak plate suggests that the sample came from a pure culture.
(left) http://www.tankonyvtar.hu/hu/tartalom/tamop412A/2011-0073_practical_microbiology/ch11s03.html
(right) http://faculty.ccbcmd.edu/courses/bio141/labmanua/lab5/M.luteus_isol_colonies.html