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Lab
03 MICROBIAL CULTURES TECHNIQUES
BTP 1213
BIOLOGY FOR ENGINEERS
Lab Objectives
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.
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, non-
corroding 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.
BTP1213 BIOLOGY FOR ENGINEERS
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.
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.
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.
BTP1213 BIOLOGY FOR ENGINEERS
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.
Figure 2.3 : Transferring Technique – stab technique in agar deep (a),(b) and (c) and
streaking technique in slant agar (d)
BTP1213 BIOLOGY FOR ENGINEERS
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.