Lab on pure culture techniques PDF

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BIOL 2026 - Exercise 7

Pure Culture Techniques

Introduction Bacteria are commonly found in mixed populations, in environments such as food, soil, water, and a variety of body organs. Under these conditions it is impossible to study the characteristics of a particular species. Therefore, pure cultures must be obtained. A pure culture contains only one species of organism. In this exercise, the streak-plate technique and the pour-plate technique will be used to separate out bacteria from a mixed culture. The principle of both techniques is to dilute or separate a mixture of bacteria so that individual cells can form single and visible groups of bacteria, called colonies. Each colony ideally arises from a single bacterium and includes all of its descendants. A sample of cells can be removed from a colony and cultivated to obtain a pure culture. The characteristics of bacterial colonies will be studied in this exercise. They indicate the genetics of the bacterium and serve as clues to various bacterial species. Colony characteristics are one of the criteria used in the identification of an unknown bacterium. A. Dilution using the streak-plate technique This technique is a rapid and inexpensive method used to separate bacteria from a mixed population. It requires an agar plate containing a growth medium that allows excellent distribution of bacterial colonies. In this technique, a dense, mixed culture is diluted out with each successive set of streaks, so that eventually single cells are deposited onto the agar. These individual cells grow and form single colonies that can be sampled and further studied. For these reasons, this is a standard technique used in many research laboratories, industry, and clinics. Materials Mixed broth culture containing Escherichia coli, Bacillus subtilis, and Staphylococcus epidermidis Tryptic soy agar (TSA) plate Demonstration plate inoculated from this broth showing colonies isolated by good streaking technique Procedures 1 week 1. Before you begin, listen carefully to the instructor as he/she demonstrates the streaking technique and familiarize yourself with the technique shown in Figure 1. Label the TSA plate (always on the BOTTOM of the plate around the perimeter) with your Group, initials, culture used, and exercise number/part. Place it lid-side down on the bench top, and vortex the mixed broth. Allow the aerosols in the tube to settle for 10-15 seconds before proceeding. 2. Using aseptic technique, sample the mixed broth with a sterilized inoculating loop. With the other hand, pick up the bottom (agar portion) of the inverted agar plate and inoculate it with the first set of streaks by making 5 streaks close to and parallel to each other (Figure 1). Hold the loop flat on the agar while streaking, not just on its edge. 3. Replace the plate into its lid and turn it clockwise (if you are right-handed) 45° on the bench top. 4. Flame the loop and pick up the bottom of the plate. Cool the loop by stabbing it once into the periphery of the agar at a spot that has not been inoculated – the area just before the 1 set of streaks is a good st

st

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BIOL 2026 - Exercise 7

Pure Culture Techniques

place (see the “X” in Figure 1). You may hear a sizzle. Pass the loop through the 1 set of streaks once and then make 4 additional and parallel streaks, as shown. Make sure that the parallel streaks do not touch each other. Replace the plate into its lid and turn it again 45° on the bench top. Flame the loop again and cool it as you did before in step 4. Pass the loop through the 2 set of streaks twice, and then make 3 additional parallel streaks. Replace the plate into its lid and turn it again 45° on the bench top. Do NOT flame the loop this time. Finally, pass the loop through the 3 set of streaks once, and, without lifting the loop from the agar, zig-zag across the remaining uninoculated portion of the agar towards, but not touching, the initial set of streaks. Use as much of the agar surface as possible. Replace the plate into its lid and sterilize the loop. st

5. 6. 7. 8.

9.

nd

rd

flame Streak once through previous set of streaks

1

2

flame

X

Streak twice through previous set of streaks

3 No flame Streak once through previous set of streaks

4

Figure 1.

The dilution streak-plate technique

10. Place the inoculated plate in its inverted (lid-side down) position onto the designated tray to be incubated at 37°C for 24 hours. The plate is inverted so that any moisture that collects on the lid will not fall onto the agar. Such moisture would cause the cells to wash together (become confluent) and not produce individual colonies. The instructor will refrigerate the plate at the end of the incubation period until the next lab session to preserve the bacterial growth results and to prevent dehydration of the medium.

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Pure Culture Techniques

11. In the Results section, make a drawing of the demonstration plate showing the separated, isolated colonies achieved using good dilution streaking technique from the mixed broth culture. This is what you are striving for when streaking your plate for isolation of colonies. 12. Prepare a Gram-stain of the original broth culture containing the 3 different bacteria and record your results. 2 week 1. Observe the agar plate that you inoculated last week and look for isolated and separated colonies, as shown in Figure 2. nd

confluent growth

miniscule colonies (may be confluent) tiny colonies

isolated colonies Figure 2 Bacterial colonies isolated on an agar plate using a good streaking technique

2. Describe the colonial morphology of the different colony types using Table 1 and Figure 3. 3. Make a drawing on page 7-7 of your agar plate. 4. Prepare air-dried, heat-fixed smears and Gram stains of each different colony type from well-isolated colonies in order to determine the cellular characteristics of each type, and complete the required results.

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BIOL 2026 - Exercise 7

Table 1.

Pure Culture Techniques

General growth/colonial characteristics on agar media, with their descriptions

1. Size: diameter in mm 2. Form (viewed from the top): punctiform, circular, irregular, filamentous, rhizoid, tapered 3. Elevation (viewed from the side): flat, raised, convex, pulvinate, umbonate, crateriform 4. Margin (edge of colony): entire, undulate, lobate, erose, filamentous, curled 5. Colour! 6. Density: opaque, translucent, transparent 7. Surface: shiny or dull, smooth or rough 8. Consistency: butyrous – mucoid –

consistency of soft butter clings to loop with which it is touched and strings away from colony when loop is withdrawn friable – delicate; when touched with a loop it crumbles into several pieces; the whole colony may move a short distance before this happens membranous – when touched with a loop, it sticks to the medium or the entire colony moves without crumbling into pieces

Form (viewed from the top)

Elevation (viewed from the side)

Margin (view edge of colony from the top or the bottom)

Figure 3.

punctiform

irregular

circular

rhizoid

filamentous

tapered

flat

pulvinate

raised

umbonate

convex

crateriform

entire

erose

undulate

filamentous

lobate

curled

Gross characteristics of bacterial colonies on agar media (Note: the figure is not to scale)

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BIOL 2026 - Exercise 7

Pure Culture Techniques

B. Dilution using the pour-plate technique In this technique, a mixed population of bacteria is progressively diluted using three tubes of a molten nutrient agar. Each tube is then mixed, the contents are poured into a petri dish, and placed into an incubator. This technique must be performed quickly so that the agar does not solidify in the tube. Agar melts at approximately 100°C and solidifies around 45°C. Materials Mixed broth culture, the same one as used in Part A 3 tubes of molten TSA, maintained as liquid in a 50°C water bath 3 sterile petri dishes (plates)

Procedures 1 week 1. Your instructor will demonstrate the technique (Figure 4) so that you know what to do to prevent the agar from solidifying in the tubes. Label the bottoms of the 3 petri dishes as 1, 2, and 3, respectively, along with your Group (A, B, …), and your initials. st

1 loopful

1 loopful

1 loopful

molten TSA

mixed broth

molten TSA

pour (1)

pour (2)

Figure 4.

molten TSA

pour (3)

The pour-plate technique

2. Vortex the mixed broth culture and allow about 10 seconds for any aerosols inside the tube to settle. Using aseptic technique, transfer a loopful of the mixed broth to the first tube of molten TSA and into the centre of the medium. Move the loop back and forth a few times to dislodge the sample from the loop. Remove the loop and flame it. Mix the contents of the tube by rolling it between the palms of both hands a few times for about 10 seconds. This mixes the contents but does not create bubbles. Vortexing would create a lot of bubbles. 3. Flame the loop and allow it to cool for a few seconds. As before, aseptically transfer a loopful from the first tube of molten agar that you mixed to a second tube of molten agar, sterilize the loop and mix the contents of the tube by rolling it between the palms of the hands.

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BIOL 2026 - Exercise 7

Pure Culture Techniques

4. Similarly, using aseptic technique, inoculate a third tube of molten agar with a sample from the second agar tube. Mix as before. 5. Carefully remove the cap of agar tube 1 and flame its mouth. Using the hand that is holding the cap, slightly lift the lid of petri dish 1 and pour all of the contents of tube 1 into the dish. 6. Replace the lid onto the petri dish and immediately and gently move the dish in a circular motion on the bench top to spread the agar evenly over the entire surface of the bottom of the dish, being careful not to slosh the agar over the edge of the plate. 7. Repeat steps 5 and 6 for tubes 2 and 3 with petri dishes 2 and 3, respectively. 6. Allow the agar to solidify in the dishes on the bench top (approximately 10 to 15 minutes). 7. Invert the plates, stack them together, and place them onto the designated tray on which they will be incubated at 37°C for 24 hours. The instructor will then refrigerate the plates until the next lab period.

2 week 1. In the Results section, make sketches of the growth for all 3 of the pour plates. Examine the isolated colonies of your plates. Colonies should be visible on the surface of the agar and within the agar. Record your observations. 2. Compare the number and variety of colonies obtained with the pour-plate technique to those obtained with the streak-plate technique. nd

Questions 1. List several advantages of the streak-plate technique compared to the pour-plate technique. Give several reasons why the pour-plate would be preferred instead of the streak-plate. 2. Why is it a good idea to incubate inoculated agar plates in an inverted position? 3. What changes could be made in the isolation technique if moulds were to be isolated from a mixed culture? 4. During the pour-plate technique, a hot loop was accidently dipped into the first molten agar tube to obtain bacteria for transfer to the second agar tube. What would be the result? 5. Explain how isolation techniques can be useful to determine the purity of a bacterial broth culture.

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Pure Culture Techniques

Results A. Observations of your Gram stain of the original mixed culture, and the streak plates 1 week st

How many different types of cells (morphology and Gram reaction) do you see in your Gram stain of the original mixed culture?

↑ Gram stain of the original mixed culture (1000X) 1 week

2nd week

st

Demonstration streak plate

Your streak plate

↑ Drawings of growth of the mixed population of Escherichia coli, Bacillus subtilis, and Staphylococcus epidermidis on the agar plates 2 week nd

Observations of the colony characteristics of your streak plate: Colony characteristics Bacterium

Abundance* size (mm) colour

form

elevation

margin

other**

E. coli B. subtilis S. epidermidis * Use a scale of 0 to 3 when estimating the abundance (number) of each different coloniy type, with 3 representing a very great abundance and 0 being no growth. **You can add other colonial characteristics, such as surface (e.g., shiny, dull), density (e.g., opaque, translucent), etc. (see Table 1).

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BIOL 2026 - Exercise 7

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Observations of your Gram stains of each colony type:

E. coli Gram ____ 1000X

B. subtilis Gram ____ 1000X

S. epidermidis Gram ____ 1000X

Based on your observations of E. coli and S. epidermidis that you Gram stained in Exercise 5 (Gram Stain) and the Gram stains that you did in this exercise, fill in the Gram reaction for each bacterium, including the third one, B. subtilis. Include drawings of the cells and their arrangements.

B. Observations from the pour plates

1 dilution plate st

2 dilution plate nd

3 dilution plate rd

↑ Drawings of the growth results for the 3 pour plates Do the sub-surface colonies differ in appearance to surface colonies? If yes, how?

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