Any discrepancy between the pH value on the control unit and
the separate pH meter can be adjusted on the set span of the
pH calibration on the control unit. Do not mix up probes.
19. BSL-1 and BSL-2 items could be heat-fixed. However, care
should be taken as this process can cause aerosolization of
viable cells, so this should be done inside a BSC. If this is a
concern, the methanol treatment is recommended. For BSL-3
items, fixing should be performed only with methanol.
Methanol-treated slides are known to inactivate the cells and
can be removed from the BSC for staining.
20. Users who are unfamiliar or inexperienced with Gram staining
should use positive and negative controls, either on commercially available slides, like BD BBL Gram slides which can hold
up to nine samples on a single slide or on slides prepared and
staining at the same time. Most labs use Staphylococcus aureus
and Escherichia coli as Gram-positive and Gram-negative staining controls, respectively.
21. AFB should look dark red to pink (these are the cells stained by
the Carbolfuschin and have not been decolorized by the decolorizer) (see Fig. 1). Non-AFB should look green to blue-green
(these are the decolorized cells that were stained with the
brilliant green counterstain).
22. Do not allow potassium permanganate to act over 2 min, or it
might quench the fluorescence of AFB.
23. Gently pass the M. leprae suspension through 27 G needle
couple of times to ensure even single-cell distribution. Avoid
frothing.
24. Carefully remove excess gelatin phenol from the slide.
25. If there are too many bacilli per field in the lower dilution
(1:100), then do not count that dilution and move to the
next higher dilution.
26. TSA is used to identify any contaminants as this media does not
contain any inhibitor compound (Middlebrook contains malachite green). However, it is common for Mycobacteria spp. to
grow on TSA; any growth on nonselective media should be
acid-fast stained to verify the colonies are mycobacteria.
27. After an isolate or a sample has been inoculated into at least one
broth and least one agar, it should be checked regularly to
monitor growth. Especially with SGM, cultures should be
checked between 3 and 5 days to determine if the sample is
contaminated with other organisms or contains a contaminating RGM. After that, cultures should be checked weekly until
growth is visible on solid media or broth tubes display an
increase in turbidity. Growth and purity can be checked
microscopically.
52
Elizabeth Wallace et al.
the separate pH meter can be adjusted on the set span of the
pH calibration on the control unit. Do not mix up probes.
19. BSL-1 and BSL-2 items could be heat-fixed. However, care
should be taken as this process can cause aerosolization of
viable cells, so this should be done inside a BSC. If this is a
concern, the methanol treatment is recommended. For BSL-3
items, fixing should be performed only with methanol.
Methanol-treated slides are known to inactivate the cells and
can be removed from the BSC for staining.
20. Users who are unfamiliar or inexperienced with Gram staining
should use positive and negative controls, either on commercially available slides, like BD BBL Gram slides which can hold
up to nine samples on a single slide or on slides prepared and
staining at the same time. Most labs use Staphylococcus aureus
and Escherichia coli as Gram-positive and Gram-negative staining controls, respectively.
21. AFB should look dark red to pink (these are the cells stained by
the Carbolfuschin and have not been decolorized by the decolorizer) (see Fig. 1). Non-AFB should look green to blue-green
(these are the decolorized cells that were stained with the
brilliant green counterstain).
22. Do not allow potassium permanganate to act over 2 min, or it
might quench the fluorescence of AFB.
23. Gently pass the M. leprae suspension through 27 G needle
couple of times to ensure even single-cell distribution. Avoid
frothing.
24. Carefully remove excess gelatin phenol from the slide.
25. If there are too many bacilli per field in the lower dilution
(1:100), then do not count that dilution and move to the
next higher dilution.
26. TSA is used to identify any contaminants as this media does not
contain any inhibitor compound (Middlebrook contains malachite green). However, it is common for Mycobacteria spp. to
grow on TSA; any growth on nonselective media should be
acid-fast stained to verify the colonies are mycobacteria.
27. After an isolate or a sample has been inoculated into at least one
broth and least one agar, it should be checked regularly to
monitor growth. Especially with SGM, cultures should be
checked between 3 and 5 days to determine if the sample is
contaminated with other organisms or contains a contaminating RGM. After that, cultures should be checked weekly until
growth is visible on solid media or broth tubes display an
increase in turbidity. Growth and purity can be checked
microscopically.
52
Elizabeth Wallace et al.