conditions. Kinyoun staining is not recommended for AFB smear
microscopy of clinical specimens, since its sensitivity is inferior to
Ziehl-Neelsen and fluorescence staining.
Following decolorization, sputum smear is counterstained with
malachite green, or methylene blue which stains the background
material, providing a contrast color against which the red AFB can
be seen. Because some mycobacterial species have a higher content
of mycolic acid in their cell walls, sometimes the decolorizing
solutions need to be modified to avoid over-decolorizing weak
acid-fast cells. For example, M. tuberculosis and M. ulcerans are
strongly acid-fast stained, and a 3% v/v acid alcohol is used to
decolorize the smear, while weak acid-fast staining species such as
M. leprae should use 0.5–1% v/v, and this should be also combined
to different staining and decolorizing time.
The auramine-O or auramine-rhodamine dyes bind mycolic
acids and fluoresces yellow/orange when exposed to UV light,
while the background is almost black. This feature provides a
more sensitive detection of AFB, which is of great value especially
when using microscopy for diagnostic purposes. However, this
staining is not specific and other acid-fast cells (e.g., some sporozoan parasites) will fluoresce yellow, so the cellular morphology
(beaded rods) is critical in considering the stain positive. Another
downfall for this technique is that most RGMs may not appear
fluorescent [16].
1.9 Processing
of Clinical Specimens
for Mycobacterial
Strain Isolation
and Culture
In order to avoid outgrowth of non-AFB fast-growing organisms
that may be present in clinical and environmental specimens over
mycobacteria, it is necessary to eliminate non-AFB and fungi before
culturing of the specimens. However, this process can also lower
mycobacteria viability and should be avoided if the specimen is
normally sterile, such as some sterile tissues and body fluids. Samples should be concentrated to maximize the recovery of mycobacteria. Sputum and other viscous specimens apart from the
decontamination and concentration steps will require a liquefaction
step [15]. One of the most common decontaminants is sodium
hydroxide (NaOH), which also serves as a mucolytic agent; however, NaOH can affect mycobacterial viability if not used with
caution regarding the concentration and time of exposure. To
minimize a detrimental effect of NaOH, some laboratories recommend adding N-acetyl-L-cysteine (NALC) to the liquefaction and
decontamination solution. In this case, the solution requires a
lower NaOH concentration. The most commonly used combination is 0.5% NALC–2% NaOH. For heavily contaminated specimens, concentrations of up to 6% NaOH can be utilized, but the
risk of inhibiting mycobacteria is higher. There are other decontamination agents used for specific specimen types, e.g., urine,
and/or known patient diseases, e.g., CF. In the case of individuals
with CF, NTM recovery from respiratory specimens is challenging.
Culturing Mycobacteria
9
microscopy of clinical specimens, since its sensitivity is inferior to
Ziehl-Neelsen and fluorescence staining.
Following decolorization, sputum smear is counterstained with
malachite green, or methylene blue which stains the background
material, providing a contrast color against which the red AFB can
be seen. Because some mycobacterial species have a higher content
of mycolic acid in their cell walls, sometimes the decolorizing
solutions need to be modified to avoid over-decolorizing weak
acid-fast cells. For example, M. tuberculosis and M. ulcerans are
strongly acid-fast stained, and a 3% v/v acid alcohol is used to
decolorize the smear, while weak acid-fast staining species such as
M. leprae should use 0.5–1% v/v, and this should be also combined
to different staining and decolorizing time.
The auramine-O or auramine-rhodamine dyes bind mycolic
acids and fluoresces yellow/orange when exposed to UV light,
while the background is almost black. This feature provides a
more sensitive detection of AFB, which is of great value especially
when using microscopy for diagnostic purposes. However, this
staining is not specific and other acid-fast cells (e.g., some sporozoan parasites) will fluoresce yellow, so the cellular morphology
(beaded rods) is critical in considering the stain positive. Another
downfall for this technique is that most RGMs may not appear
fluorescent [16].
1.9 Processing
of Clinical Specimens
for Mycobacterial
Strain Isolation
and Culture
In order to avoid outgrowth of non-AFB fast-growing organisms
that may be present in clinical and environmental specimens over
mycobacteria, it is necessary to eliminate non-AFB and fungi before
culturing of the specimens. However, this process can also lower
mycobacteria viability and should be avoided if the specimen is
normally sterile, such as some sterile tissues and body fluids. Samples should be concentrated to maximize the recovery of mycobacteria. Sputum and other viscous specimens apart from the
decontamination and concentration steps will require a liquefaction
step [15]. One of the most common decontaminants is sodium
hydroxide (NaOH), which also serves as a mucolytic agent; however, NaOH can affect mycobacterial viability if not used with
caution regarding the concentration and time of exposure. To
minimize a detrimental effect of NaOH, some laboratories recommend adding N-acetyl-L-cysteine (NALC) to the liquefaction and
decontamination solution. In this case, the solution requires a
lower NaOH concentration. The most commonly used combination is 0.5% NALC–2% NaOH. For heavily contaminated specimens, concentrations of up to 6% NaOH can be utilized, but the
risk of inhibiting mycobacteria is higher. There are other decontamination agents used for specific specimen types, e.g., urine,
and/or known patient diseases, e.g., CF. In the case of individuals
with CF, NTM recovery from respiratory specimens is challenging.
Culturing Mycobacteria
9