Pure CultureTechniques

acteria usually exist in mixed populations in soil, water, andsome parts of the human body. It is not feasible to identify orstudy the characteristics of a particular species when it is mixed

with other species, and, therefore, a pure culture must be obtained inpreparation for further work. As the name implies, a pure culture containsonly a single species of organism.

In this exercise, the streak plate technique and the pour plate tech-nique will be used to separate and isolate the bacteria in a mixed population.The fundamental principle underlying both techniques is that a mixture ofbacteria is gradually thinned out in a growth medium, giving individualorganisms room to form separate masses of bacteria called colonies. Pre-sumably, each colony arises from the growth and reproduction of a singlebacterium, and therefore all the cells in the colony are genetically identical.A sample of the cells may then be selected and subcultured to form a pureculture.

Bacterial colony characteristics also will be studied in this exer-cise. These characteristics reflect the genetic composition of a bacterium andserve as markers for different bacterial species. The laboratory microbiol-ogist often uses colony characteristics as one criterion in the identificationof an unknown bacterium.

Streak Plate Technique

The quadrant streak plate technique is a relatively inexpensive and rapidmethod for separating bacteria in a mixed population of high cell density. Itrequires only a single plate of growth medium and, with practice, it yieldsexcellent distribution of bacterial colonies. For these reasons, the technique isstandard practice in many research, industrial, and clinical laboratories.

pecial Materials

• Mixed population of bacteria in broth culture• Nutrient agar plates or materials for their preparation

S

A.

B

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PURPOSE: to perform thestreak plate technique forisolating a single species inpure culture.

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rocedure

1. Select a nutrient agar plate or, at the direction of the instructor, pour aplate of the medium using a Petri dish and melted nutrient agar. With a waxpencil or felt marker, label the bottom side of the plate with your name, thedate, and the designation “streak plate.” Obtain a broth culture containinga mixed population of bacteria. Before beginning the streak plate tech-nique, read through steps 2 through 4 and review Figure 2.1 to familiarizeyourself with the technique.

2. Aseptically obtain a loopful of the mixed population, and lightly streak it sev-eral times along one area (quadrant) of the plate, as shown in Figure 2.1A.Try not to cut into the agar surface and avoid airborne contamination by lift-ing the lid of the Petri dish only enough to permit entry of the loop.

3. Replace the lid. Sterilize the loop to destroy any remaining bacteria. Toensure that it is cool, touch the loop to the center of the plate or between theagar and the edge of the plate. Pass the loop one time across the previousstreaks to pick up some bacteria, and continue streaking into a secondarea of the plate, as illustrated in Figure 2.1B. Replace the lid.

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12 2 P U R E C U L T U R E T E C H N I Q U E S

Quick ProcedureStreak Plate Technique

1. Streak a loopful of bacteria in one area ofan agar plate.

2. Flame the loop andstreak some bacteriafrom the first area into asecond area.

3. Flame the loop andstreak some bacteriafrom the second areainto a third area.

4. Flame the loop andstreak some bacteriafrom the third area intoa fourth area. Incubate.

Quadrant 1

Quadrant 1

2

3

Quadrant 1

24

3

Quadrant 1

2

Third streak

Fourth streak

Second streak

Firststreak

A

C

B

D

F I G U R E 2 . 1The streak plate isolation technique.

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P U R E C U L T U R E T E C H N I Q U E S 2 13

4. Sterilize the loop as before, then be certain that it is cool. To pick up somebacteria, pass the loop one time through the second area, and continuestreaking into a third area (Figure 2.1C). Replace the lid.

5. Sterilize the loop as before, and streak some bacteria from the third area of theplate into the fourth area (Figure 2.1D). Be sure to use up the remainingspace on the plate. Replace the lid. Sterilize the loop.

6. To obtain practice, repeat the streak plate technique using additional platesand different mixed populations. The instructor may recommend alter-native streaking methods.

7. Additional practice can be obtained by attempting to isolate bacteria onselective media. For example, streptococci can be cultivated on mitis sali-varius agar using a swab of material from the gingival (tooth-gum) crevice(Exercise 16); staphylococci can be cultivated on mannitol salt agar usingmaterial swabbed from the nose cavity (Exercise 18); and intestinal bacteriacan be cultivated on MacConkey agar and EMB agar using a fecal sus-pension as a source (Exercise 19).

8. Invert all the plates and incubate them for 24–48 hours at the appropriatetemperature. The plates are inverted so that moisture accumulates on thelid rather than on the agar surface, where it may cause colonies to runtogether. After incubation, refrigerate the plates in the inverted position untilthe next laboratory session to preserve the bacterial growth and preventdrying of the medium.

9. Examine the plates for well-isolated and separated colonies, as shown in Figure 2.2, and enter a representation of a good streak plate in the Resultssection. Add appropriate labels and brief explanations to produce a “talk-ing picture.” Describe several isolated colonies by numbering the coloniesand noting their size, color, and characteristics, with reference to the stan-dard terminology provided in Figure 2.3. Size may be determined by mea-suring the colony diameter in millimeters if rulers are available. Recordyour observations in Table 2.1 in the Results section.

10. At the direction of the instructor, use a bacteriological needle or loop toselect samples from various colonies, and inoculate nutrient agar slantsto obtain pure cultures. Stained smears may also be made from the coloniesto determine the morphological characteristics of the organisms.

Confluent growth

Tiny colonies

Isolated colonies

4

3

Quadrant 1

2

F I G U R E 2 . 2Isolated colonies of bacteria on a well-executed streakplate.

!Never touch the bacterialcolonies on a plate with yourfingers. Each colony containsmillions of live organisms.

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Pour Plate Technique

In the pour plate technique, a mixed population of bacteria is progres-sively diluted in three “deep” tubes of liquid nutrient agar. The medium isthen poured into Petri dishes for incubation. Colonies of bacteria subse-quently appear on the surface as well as within the medium. Once it isstarted, the technique must be performed without delay because the liquidagar solidifies quickly in the tubes.

pecial Materials

• Mixed populations of bacteria in broth culture• Deep tubes of liquid nutrient agar• Sterile Petri dishes

rocedure

1. The pour plate technique must be performed rapidly to prevent prematurehardening of the agar medium in the test tubes. Therefore, it is well toreview steps 2 through 6 before beginning the procedure. Refer also toFigure 2.4 to obtain a thorough understanding of what you will be doingbefore starting your work.

P

S

B.

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Form Elevation

Margin

Circular

Raised

Entire

Irregular

Convex

Undulate

Filamentous

Umbonate

Filamentous

Rhizoid

Crateriform

Curled

Punctiform

Flat

Lobate

F I G U R E 2 . 3Standard terminology for bacterial colony characteristics.

PURPOSE: to perform thepour plate technique for iso-lating a single species in pureculture.

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2. Obtain three sterile Petri dishes and label the bottom sides with yourname, the date, and the designations “Pour Plate #1,” “Pour Plate #2,”and “Pour Plate #3.” The instructor will explain the preparation of thenutrient agar deep tubes and how they are melted and maintained atapproximately 45º–50º C to prevent solidification. Obtain three tubes for thetechnique. A mixed population of bacteria should also be available.

3. Aseptically obtain a loopful of the mixed population (Figure 2.4A). Inocu-late it into the first melted agar deep tube by placing the loop below the sur-face and shaking the loop lightly. Resterilize the loop and replace it in itsholder. Now mix the contents of the tube by rolling it between the palmsof the hands or by vigorously tapping its bottom with the index finger.

4. Sterilize the loop and be certain that it is cool. Obtain one loopful of liquidagar from the first deep tube and inoculate it into the second deep tube(Figure 2.4B). Sterilize the loop, then mix the tube contents well by rollingor tapping the tube.

5. Sterilize the loop, ensure that it is cool, and then obtain one loopful of liq-uid agar from the second deep tube and inoculate it into the third deep tube(Figure 2.4C). Mix the contents well.

6. Aseptically pour the contents of each tube into the designated Petri dish (Figure 2.4D–F), being sure to flame the neck of the tube after removing thecap. Gently rotate the dishes in a wide arc on the laboratory desk to dis-tribute the medium evenly over the bottom of the plates.

7. Allow the agar to solidify for several minutes, then incubate the plates inthe inverted position, as outlined in Part A, step 8.

8. Examine the plates for well-isolated colonies. Surface as well as subsurfacecolonies should be visible. Enter a well-labeled and explained represen-tation of the plate series in the appropriate space in the Results section, anddescribe the colonies as outlined in Part A, step 9 in Table 2.1. Compare the

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Quick ProcedurePour Plate Technique

1. Inoculate a loopful ofbacteria to a meltedagar deep tube.

2. Transfer a loopful fromthe first deep tube to asecond deep tube.

3. Transfer a loopful fromthe second deep tube toa third deep tube.

4. Pour the three deeptubes of agar into Petridishes. Incubate.

A

D E F

B CAsepticallytransferone loopful from the sample totube #1.

Pour the contentsof tube #1 into asterile Petri dish.

Pour the contentsof tube #2 into asterile Petri dish.

Pour the contentsof tube #3 into asterile Petri dish.

Aseptically transfer one loopful from tube #1 totube #2.

Aseptically transfer one loopful from tube #2 totube #3.

Mixed populations sample

#1

Plate #1

#2

Plate #2

#3

Plate #3

F I G U R E 2 . 4The pour plate isolation technique.

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number and variety of colonies appearing in this technique with thoseappearing in the streak plate technique as part of your notes. At the instruc-tor’s direction, transfer samples from the plate to nutrient agar slants forpure culture cultivation, and prepare smears for staining. Subsurfacecolonies may be reached by applying a warm inoculating needle to melt theagar down to the level of the colony.

uestions

1. Explain several advantages of the streak plate technique over the pourplate technique, and several reasons why the pour plate technique mightbe preferred to the streak plate technique.

2. Predict the outcome of incubating inoculated streak and pour plates inthe upright rather than the inverted position.

3. What variations might be made in the isolation techniques if molds wereto be isolated from a mixed culture?

4. While performing the pour plate technique, a student inadvertently dippeda hot loop into agar tube #1 to obtain bacteria for agar tube #2. What willresult?

5. Explain how isolation methods might be useful in determining the purityof a bacterial culture.

Q

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P U R E C U L T U R E T E C H N I Q U E S 2 17

Name

Date Section

Exercise Results

Pure Culture Techniques

A. Streak Plate Technique

2

Source: ________________________

Streak Plate #1 Streak Plate #2

Source: ________________________

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Table 2.1. Summary of Colony Characteristics

Colony Number Colony Characteristicsand Source Size Color Form Elevation Margin

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B. Pour Plate Technique

Pour Plate #1 Pour Plate #2

Pour Plate #3

Source: ________________________

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Source: ______________________ ______________________ ______________________

Magnif.: ______________________ ______________________ ______________________

Observations and Conclusions:

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