Lab 03 Microbial Cultures Techniques(1).docx

FACULTY OF ENGINEERINGTECHNOLOGY

Lab 03 MICROBIAL CULTURES TECHNIQUES BTP 1213 BIOLOGY FOR ENGINEERS Lab Objectives By the end of this lab, students should be able to: 1. To perform aseptic transfers of bacteria culture using inoculating loop and inoculating needle. 2. To be able to differentiate between agar slants, agar deep, agar plates and nutrient broths.

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BTP1213 BIOLOGY FOR ENGINEERS

EXPERIMENT 3: MICROBIAL CULTURES TECHNIQUES

1. To perform aseptic transfers of bacteria culture using inoculating loop and inoculating needle. 2. To be able to differentiate between agar slants, agar deep, agar plates and nutrient broths.

In the laboratory, bacteria must be cultured in order to facilitate identification and to examine their growth and metabolism. Bacteria are inoculated, or introduced, into various forms of culture media in order to keep them alive and to study their growth. Inoculations must be done without introducing unwanted microbes, or contaminants, into the media. Aseptic technique is used in microbiology to exclude contaminants.

Broth cultures provide large numbers of bacteria in a small space and are easily transported. Agar slants are test tubes containing solid culture media that were left at an angle while the agar solidified. Agar slants, like agar plates, provide a solid growth surface, but slants are easier to store and transport than agar plates. Agar is allowed to solidify in the bottom of a tube to make an agar deep. Deeps are often to grow bacteria that prefer less oxygen than is present on the surface of the medium.

Transfer and inoculation are usually performed with a sterile, heat-resistant, noncorroding Nichrome wire attached to an insulated handle. When the end of the wire is bent into a loop, it is called an inoculating loop. When straight, it is called an inoculating needle. For special purposes, cultures may also be transferred with sterile cotton swabs, pipettes, glass rods, or syringes.

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Inoculating loop, inoculating needle Nutrient agar slants, nutrient agar deep, nutrient agar plates, sterile nutrient broths Escherichia coli broth in universal bottle Escherichia coli on agar plate Pseudomonas sp broth in universal bottle

: Weigh out 8g of nutrient broth powder. Add to 1L of distilled or deionized water in a 1L Schott bottle. Dissolve the powder completely in the water. Sterile at 121°C for 20 minutes.

: Weigh out 23g of nutrient agar powder. Add to 1L of distilled or deionized water in a 1L Schott bottle. Sterile at 121°C for 20 minutes. Cool to 50°C.

: Pour 15-20mL of a warm sterile nutrient agar per petri plate. Allow the nutrient agar to harden.

: Pipette 5mL of a warm sterile nutrient agar into test tube or universal bottle. Place the cap loosely. Place the test tube or universal bottle in a bend position. Allow the nutrient agar to harden in this position.

: Pipette 5mL of a warm sterile nutrient agar into test tube or universal bottle. Place the cap loosely. Place the test tube or universal bottle in up-right position until the molten agar harden.

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1.

Turn on the flame of Bunsen burner. Flame the inoculation loop to redness. Allow it to cool. Aseptically obtain a loopful of one broth culture.

2.

To streak the plate, lift one edge of petri plate cover, and streak the first sector by making as many streaks as possible without overlapping previous streaks. Do not gouge the agar while streaking the plate. Hold the loop as you would hold a pencil or paintbrush, and gently touch the surface of the agar.

3.

Flame your loop and let it cool. Turn the plate so the next sector is on top. Streak through one area of the first sector, then streak a few times away from the first sector.

4.

Flame your loop, turn the plate again, and streak through one area of the second sector. Then streak the third sector.

5.

Flame your loop, streak through one area of the third sector, and then streak the remaining area of the agar surface, being careful not to make additional contact with any streaks in the previous sections. Flame your loop before setting it down.

6.

Label each of petri plate and incubate at 30oC for 24h. Incubate the plates in an inverted position.

Figure 2.1: Quadrant Streak for Streaking Method.

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1.

Turn on the flame of Bunsen burner. Flame the inoculation loop to redness. Allow it to cool. Aseptically obtain a loopful of one broth culture.

2.

Inoculate the agar slant by moving the loop gently across the agar surface from the bottom of the slant to the top.

3.

Flame the mouth of the test tube or universal bottle and replace the cap.

4.

Reflame the loop and let it cool.

5.

Incubate the agar slant at 30oC for 24h.

Figure 2.2: Aseptic Technique for Bacterial Removal and Subculturing

1.

Turn on the flame of Bunsen burner. Flame the inoculation loop to redness. Allow it to cool.

2.

Hold the inoculating loop in your dominant hand. Aseptically obtain a loopful of one broth culture from the plate.

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3.

Using the other hand and the dominant hand’s small finger, open the broth bottle cap and flame the mouth of the bottle to avoid contamination and recap it.

4.

Using your dominant hand with the inoculating loop, inoculate the broth by immersing the loop into the bottle. Flame the mouth of the bottle and replace the cap.

5.

Flame your loop and let it cool.

6.

Label the broth bottle and incubate at 30oC for 24h.

1.

Turn on the flame of Bunsen burner.

2.

Hold theinoculating needle in your dominant hand. Flame the inoculating needle until it is red and let it cool. Aseptically obtain a loopful of bacteria culture from the agar plate.

3.

Using the other hand and the dominant hand’s small finger, open the test tube cap and flame the mouth of the bottle to avoid contamination and recap it.

4.

Again, using the other hand and the dominant hand’s small finger, open the test tube cap and flame the mouth of the bottle to avoid contamination.

5.

Using your dominant hand with the inoculating needle, inoculate the semisolid agar deep by plunging the needle straight down the middle of the deep and then pulling it through the same stab. Flame the mouth of the test tube and replace the cap.

6.

Flame your needle and let it cool.

7.

Label the test tube and incubate at 30oC for 24h.

Figure 2.3 : Transferring Technique – stab technique in agar deep (a),(b) and (c) and streaking technique in slant agar (d)

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1.

Sketch the appearance of each culture on agar plate, agar slant and agar deep. Observe any pigmentation on each agar.

2.

Sketch and describe the nutrient broth cultures in term of turbidity and pigmentation.