3. M17 broth can also be stored at room temperature if used in a
short period of time. Keeping it at room temperature also helps
identifying contamination of the medium.
4. Remove the petri dishes from the refrigerator and leave it on
the bench until it reaches room temperature before inoculating
the bacteria.
5. To count the number of CFU, choose the dilution factor in
which between 30 and 300 CFU grow to make it easier to
count and reduce mistakes.
6. The colony-forming unit (CFU) is a measure of viable bacterial
cells, the results are given in CFU/mL (colony-forming units
per milliliter). For the CFU measurement, perform serial dilution (10
À6 , 10
À7 , 10
À8 , or more if necessary) or you will not be
able to count the colonies.
7. The dilution is made to the lag phase so the dose will be
prepared on the exponential phase.
8. For the bacterial growth curve, an initial of OD 600 ¼ 0.04 is
used because this OD 600 corresponds to the bacteria during the
lag phase.
9. Each dose is equivalent to 2 mL of bacterial culture.
10. The prepared doses can be used up to 5 days after production if
stored at À80
C; after this time, the doses start to lose
viability.
Fig. 8 Nasal administration. With the animal restrained, place the dose to be
administered, with the help of a pipette, above the nares of the animal letting it
be absorbed before administering the next dose
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short period of time. Keeping it at room temperature also helps
identifying contamination of the medium.
4. Remove the petri dishes from the refrigerator and leave it on
the bench until it reaches room temperature before inoculating
the bacteria.
5. To count the number of CFU, choose the dilution factor in
which between 30 and 300 CFU grow to make it easier to
count and reduce mistakes.
6. The colony-forming unit (CFU) is a measure of viable bacterial
cells, the results are given in CFU/mL (colony-forming units
per milliliter). For the CFU measurement, perform serial dilution (10
À6 , 10
À7 , 10
À8 , or more if necessary) or you will not be
able to count the colonies.
7. The dilution is made to the lag phase so the dose will be
prepared on the exponential phase.
8. For the bacterial growth curve, an initial of OD 600 ¼ 0.04 is
used because this OD 600 corresponds to the bacteria during the
lag phase.
9. Each dose is equivalent to 2 mL of bacterial culture.
10. The prepared doses can be used up to 5 days after production if
stored at À80
C; after this time, the doses start to lose
viability.
Fig. 8 Nasal administration. With the animal restrained, place the dose to be
administered, with the help of a pipette, above the nares of the animal letting it
be absorbed before administering the next dose
458
Viviane Lima Batista et al.