Journal of Microbiological Methods 91 (2012) 128–132

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Journal of Microbiological Methods

journal homepage: www.elsevier.com/locate/jmicmeth

A modified acid-fast staining method for rapid detection of

Mycobacterium tuberculosis
Dan Zhao a, b,⁎, Xiao-Meng Yang c, Qian-Yu Chen b, Xing-Shu Zhang b, Chun-Jiang Guo b, Xiao-Yan Che a
a
Second Clinical Medical College of Southern Medical University, Guangzhou, 510280, China
b
Department of Laboratory, Luohu Chronic Disease Prevention and Cure Hospital, Shenzhen, 518023, China
c
Department of Genetics Laboratory, Luohu Maternity and Childcare Hospital, Shenzhen, 518019, China

a r t i c l e i n f o a b s t r a c t

Article history: A modified acid-fast staining method was developed for rapid detection of Mycobacterium tuberculosis and its L
Received 24 March 2012 forms, wherein carbol fuchsin and dioxogen were mixed into the sputum smear. With this method, the dyeing
Received in revised form 25 July 2012 time is shortened and heating is not required. The sensitivity, specificity, positive predictive value, negative pre-
Accepted 25 July 2012
dictive value, positive rate, and diagnostic efficiency of the new method were compared to those obtained by PCR
Available online 11 August 2012
using 50 clinical samples. Further, 468 clinical samples were analyzed using the new method, the modified inten-
Keywords:
sified Kinyoun (IK) acid-fast staining method, and the traditional Ziehl–Neelsen acid-fast staining method.
Mycobacterium tuberculosis Differences among the positive detection rates of the three methods were analyzed using Student's t-test, and
Modified acid-fast staining method no significant differences were found between the new method and the modified IK acid-fast staining method,
Carbol fuchsin while the rates of both these methods were higher than that of the traditional acid-fast staining method. Addi-
Dioxogen tionally, the dyeing time in the new method was markedly less than that in the modified IK acid-fast staining
method (5 min and 24 h, respectively).
© 2012 Elsevier B.V. All rights reserved.

1. Introduction MTB and L-MTB can be identified by microscopic examination, cul-

Mycobacterium tuberculosis (MTB) can form cell wall-deficient
mutants (L forms) easily, and M. tuberculosis L forms (L-MTB) are
able to survive under harsh environmental conditions. L-MTB can sur-
vive over long periods in vivo and can be isolated and cultivated from
blood samples of patients with active sarcoidosis (Almenoff et al.,
1996). The biological characteristics, pathogenicity, and drug suscep-
tibility of L-MTB differ from those of MTB, and these differences might
be important factors underlying tardy progression, aggravation, re-
currence, and drug resistance in tuberculosis (Seiler et al., 2003; Lu
et al., 2010). Approximately 70% of active pulmonary tuberculosis cases
are reported to be smear and culture negative (Chinese Society of
Tuberculosis, 2001). However, the L-MTB positive rate exceeds 30% for
sputum specimens from smear- and culture-negative pulmonary tuber-
culosis cases, and the L-MTB positive rate exceeds 50% for sputum spec-
imens from multidrug-resistant tuberculosis cases (Dorozhkova et al.,
1990; Bobchenok et al., 2002; Cui et al., 2004). Thus, a fast and conve-
nient method to detect MTB and L-MTB would be useful in determining
the nature of a tuberculosis infection, selecting a treatment strategy, and
identifying preventive measures.
tivation (Rodrigues et al., 2009), immunological methods (Fernández
et al., 2011), and PCR-based methods (Vishnevskaia et al., 2001;
Fang et al., 2010). However, the last three methods may not be suit-
able for routine examinations in a primary hospital because of their
rigorous laboratory conditions and/or their relatively high cost. Micro-
scopic examination, such as that based on the traditional Ziehl–
Neelsen acid-fast staining and modified intensified Kinyoun (IK) acid-
fast staining methods, is relatively easy (Domingue, 1982; The Editorial
Board of Chinese Journal of Tuberculosis and Respiratory Diseases,
2003). However, these methods also have obvious shortcomings, for
example, colorants need to be added to the smear repeatedly, heating
is required for the Ziehl–Neelsen acid-fast staining method, and the
evaporative carbol used is harmful for the operant. Although heating is
not needed in the modified IK acid-fast staining method, its completion
requires about 24 h; moreover, quality control of the long and compli-
cated process is difficult (Cui et al., 2004). Therefore, it is necessary to
develop a rapid and convenient MTB and L-MTB staining method.
2. Materials and methods

2.1. Patients
⁎ Corresponding author at: Department of Laboratory, Luohu Chronic Disease Prevention
and Cure Hospital, Shenzhen 518023, China. Tel.: +86 755 8245 1811; fax: +86 755 8205
6724.
Four hundred and sixty-eight patients with confirmed tuberculo-
E-mail addresses: Olivia_dan@126.com (D. Zhao), sysu_microbiology@126.com sis from Luohu Chronic Disease Prevention and Cure Hospital were
(X.-M. Yang), chexiaoyan@126.com (X.-Y. Che). included in our study.

0167-7012/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.mimet.2012.07.024
 D. Zhao et al. / Journal of Microbiological Methods 91 (2012) 128–132 129

2.2. Instruments and reagents
Acid-fast staining solution was obtained from BASO Diagnostic
(Zhuhai, China). Fluorescent quantitative (FQ) PCR kits for MTB were
obtained from Da'an Gene (Guangzhou, China). Dioxogen (30%, A. P.)
was obtained from JOTAN (Zhengzhou, China). An OLYMPUS-CX31
light microscope (OLYMPUS, Japan) and an HH.CP-01.160 CO2 incuba-
tor (Boxun, Shanghai, China) were used. The real-time PCR instrument
(ABI-7300) was obtained from Applied Biosystems (Foster City, CA).
2.3. New acid-fast staining method, modified IK acid-fast staining method,
and traditional acid-fast staining method procedures
The modified IK acid-fast staining (Domingue, 1982) and traditional
Ziehl–Neelsen acid-fast staining (The Editorial Board of Chinese Journal
of Tuberculosis and Respiratory Diseases, 2003) methods were execut-
ed as reported previously. The procedural steps are presented in Fig. 1.
Our modified acid-fast staining method used different staining mate-
rials, did not require heating (Fig. 1), and took only about 5 min.
2.4. DNA extraction and real-time PCR analysis
MTB DNA was extracted from 50 sputum samples from patients
with confirmed tuberculosis by using the commercially available
M. tuberculosis (TB) fluorescent polymerase chain reaction diagnos-
tic kit (Da'an Gene, http://en.daangene.com/) according to the manu-
facturer's instructions. Extracted DNA was stored at −20 °C in extract
buffer until analysis.
MTB DNA was analyzed by using a previously reported real-time
PCR method (Zhu et al., 2008). Briefly, real-time PCR was carried

Our modified method Modified IK method Traditional method

Drop sputum onto slide Drop sputum onto slide Drop sputum onto slide

Air dry slide at room Air dry slide at room Air dry slide at room temperature,
temperature, fast heat fix slide temperature, fast heat fix heat fix slide for 10 min at 90 °C
by flame slide by flame

Flood slide with Carbol
Fuchsin, add 2 or 3 drops of
dioxogen (30%) into Carbol
Fuchsin, allow slide to stand 1 -
2 min, rinse gently with tap
water
Flood slide with Kinyoun
carbol Fuchsin in wet box
at room temperature for 24
h, rinse gently with tap
water
Flood slide with Carbol Fuchsin,
hold a flame beneath the slide until
steam appears but do not allow it
to boil, allow slide to stand in hot
solution for 3-5 min, rinse gently
with tap water

Flood slide with acid-alcohol Flood slide with acid-alcohol Flood slide with acid-alcohol (5%
(5% HCl in ethanol) (3% HCl in ethanol) HCl in ethanol)

Allow acid-alcohol to stand Allow acid-alcohol to stand Allow acid-alcohol to stand until
until no macroscopic red until no macroscopic red color no macroscopic red color on the
color on the slide, rinse on the slide, rinse gently with slide, rinse gently with tap water
gently with tap water tap water

Flood slides with methylene Flood slides with methylene Flood slide with methylene blue for
blue for 30 seconds, rinse blue for 0.5 - 2 min, rinse 1 min, rinse gently with tap water
gently with tap water gently with tap water

Allow slides to air dry before Allow slides to air dry before Allow slides to air dry before
viewing viewing viewing

Fig. 1. Procedural steps within three acid-fast staining methods. The blue column presents the steps in our modified acid-fast staining method; the pink column presents the steps in
the modified IK acid-fast staining method; and the green column presents the steps in the traditional Ziehl–Neelsen acid-fast staining method.
130 D. Zhao et al. / Journal of Microbiological Methods 91 (2012) 128–132
out by using the commercially available M. tuberculosis (TB) fluores-
cent polymerase chain reaction diagnostic kit (Da'an Gene) in a
40-μL reaction mixture containing PCR buffer, deoxynucleoside tri-
phosphates (i.e., dATP, dTTP, dCTP, and dGTP), forward and reverse
primers (5′-TCGCCCGTCTACTTGGTGTT-3′; 5′-TGATGTGGTCGTAGT
AGGTC-3′), and the fluorescence probe (5′-ACAACGCCGAATTGCG
AAGGGC-3′), along with 3 μL Taq DNA polymerase and 2 μL of the
extracted DNA sample. The thermal cycler was programmed as
follows: 1 cycle of 93 °C× 2 min, 10 cycles of 93 °C× 45 s and 55 °C×
1 min, and 30 cycles of 93 °C × 30 s and 55 °C× 45 s. The fluorescence
signals of 6-carboxy-fluorescein were acquired at the end of the third
program (i.e., after 55 °C× 45 s). Fluorescence data analysis was
conducted using SDS software (Version 1.4; Applied Biosystems). The
threshold fluorescence level, used to derive threshold cycle values,
was automatically determined by the SDS software.
2.5. Evaluation of our modified acid-fast staining method
The sensitivity, specificity, positive predictive value, negative pre-
dictive value, positive rate, and diagnostic efficiency of our modified
acid-fast staining method were evaluated by using the PCR method
as the comparison's gold standard (Vishnevskaia et al., 2001; Alli et
al., 2011; Chen et al., 2012; Zakham et al., 2012). Briefly, fifty sputum
samples from patients with confirmed tuberculosis were analyzed by
comparing results from our modified acid-fast staining method and
the PCR method (Zhu et al., 2008).
2.6. Clinical application of our modified acid-fast staining method
Four hundred and sixty-eight sputum samples from patients with
confirmed tuberculosis were smeared on individual glass slides to
form egg-shaped sputum membranes of size 10 × 20 mm (6 smears
per sample). Two of the six smears were analyzed using the traditional
acid-fast staining method; two, using the modified IK acid-fast staining
method; and two, using our modified acid-fast staining method. The
positive detection rates for MTB and L-MTB in the smears of the 468
samples treated with each of the three methods were compared.
2.7. Statistical analysis
Accuracy was defined by the statistical measures of sensitivity and
specificity. Sensitivity expressed the proportion of correctly identified
positive results, and specificity indicated the proportion of correctly
identified negative results:
Sensitivityð% Þ ¼ ðtrue positive results  100Þ
=ðtrue positive þ false negative resultsÞ;
Specificityð% Þ ¼ ðtrue negative results  100Þ
=ðtrue negative þ false positive resultsÞ;
Positive predictive valueð% Þ ¼ ðtrue positive results  100Þ
=ðtrue positive þ false positive resultsÞ;
Negative predictive valueð% Þ ¼ ðtrue negative results  100Þ
=ðtrue negative þ false negative resultsÞ:
The percentage of samples correctly identified (i.e., ((true positive
results + true negative results) × 100 / all results)) was used to indi-
cate diagnostic efficiency.
Differences between values from our modified acid-fast staining
method and the PCR method were assessed by using McNemar's chi
square test. Differences among values from our method, the modified
IK acid-fast staining method, and the traditional acid-fast staining
method were assessed by using Student's t-test. Differences yielding
probability (P) values of b 0.05 were regarded as significant.
3. Results
3.1. Micrographs of M. tuberculosis and M. tuberculosis L forms obtained
with the three acid-fast staining methods
Red stained L-MTB and MTB were observed on slides stained using
all three methods (Fig. 2).
3.2. Comparison of results from detection using our method and the PCR
method
For the 50 sputum samples from patients with definite tuberculosis,
the positive detection rate for L-MTB was 30% with the PCR method,
while it was 26% with our method. However, this difference was not
found to be significant (corrected χ2 = 0.25, P = 0.61715).
The sensitivity, specificity, positive predictive value, negative predic-
tive value, and diagnostic efficiency of our modified acid-fast staining
method were 80%, 97.1%, 92.3%, 94.4%, and 92%, respectively (Table 1).
3.3. Detection results for the three acid-fast staining methods
For the 468 sputum samples from patients with confirmed tuber-
culosis, there was no significant difference in the positive detection
rates of MTB and L-MTB between our method and the modified IK
acid-fast staining method (u=0.392, P=0.3483, u=0.155, P=0.4404,
respectively). However, the positive detection rates of MTB and L-MTB
obtained with our method were significantly higher than those obtained
with the traditional acid-fast staining method (u=3.684, Pb 0.0010, u=
3.713, Pb 0.0010, respectively); the positive detection rates of MTB and
L-MTB obtained with the modified IK acid-fast staining method were
significantly higher than those obtained with the traditional acid-fast
staining method (u=5.818, Pb 0.0010, u=4.264, Pb 0.0010, respectively;
Fig. 3).
4. Discussion
Here, we describe a new, modified acid-fast staining method, which
is convenient, specific, and sufficiently sensitive to examine sputum for
the presence of L-MTB and MTB quickly and routinely. Our method can
be easily utilized in a general tuberculosis laboratory.
High lipid content is present in the cytoderm of Mycobacterium,
comprising about 60% of the dry weight of the bacterium. In addition,
the peptidoglycan layer of the bacterium is covered by a generous
layer of mycolic acid, which can impede the penetration of colorants
into the bacterium. The traditional Ziehl–Neelsen acid-fast staining
method can be used to detect L-MTB, but the result is often negative
because L-MTB is more resistant to colorants than MTB. In our meth-
od, dioxogen was added to the smear after addition of a carbol fuchsin
solution. Dioxogen is a strong oxidant that induces lipid peroxidation
in the cytoderm. This peroxidation may damage the cytomembrane
and increase membrane permeability (Yajima et al., 2009; Pandey et
al., 2010). As a result, colorants can penetrate easily into the bacteria.
As in the modified IK acid-fast staining method, no heating is needed
in our method. In addition, our method only needs about 5 min to
complete, which is markedly shorter than the 24 h required to com-
plete the modified IK acid-fast staining method. The results obtained
using our method has satisfactory concordance with those obtained
by PCR. Moreover, the MTB and L-MTB positive detection rates of
our method were significantly higher than those of the routine acid-
fast staining method.
L-MTB presence is closely correlated with anergy in tuberculosis,
multidrug resistance, and recurrent tuberculosis, all of which are in-
volved in the drug resistance mechanism and the survival mechanism
 D. Zhao et al. / Journal of Microbiological Methods 91 (2012) 128–132 131

Fig. 2. Images of M. tuberculosis or M. tuberculosis L forms obtained by using three kinds of acid-fast staining (×1000). Catenulate M. tuberculosis L forms indicated by the arrow (A) and
slender M. tuberculosis (B) were observed on the slide stained using our modified acid-fast staining method. Slender M. tuberculosis were observed on the slide stained using the modified
IK acid-fast staining method (C) and the traditional Ziehl–Neelsen acid-fast staining method (D).

of resistant mutants in vivo (Michailova et al., 2005). Cell wall alter-
ations were observed in bacteria causing latent tuberculosis infections
in Ziehl–Neelsen staining-negative patients (Seiler et al., 2003). Inten-
sive examination of L-MTB is helpful for examining the clinical and
epidemiological significance of such infections. L-MTB of various
shapes, such as particles, spheroplasts, or thread-like L-MTB, indicate
that the L-MTB have formed under various unsuitable conditions, in
vitro and in vivo (Zhu, 2008). L-MTB pathogenicity is related to the
presence of protein, lipid, and saccharide in the cell wall and is weaker
than that of MTB because of absent or reduced antigenic components
in the cell wall. Lymphadenectasis and caseous necrosis are often
found only in L-MTB-infected patients, and typical clinical signs such
as tubercles are infrequent. As a result, L-MTB infections are often
misdiagnosed based on pathological changes, as chronic lymphadeni-
tis. In previous studies, positive sputum smear patients represent
about 64.7% of all active tuberculosis patients, while 86.3% of those
positive smear patients are infected with typical MTB, and 42.7% are
infected with L-MTB; among them, 29% are infected with MTB and
L-MTB (Dorozhkova et al., 1989, 1990). The positive rates of L-MTB
in multidrug-resistant tuberculosis cases may reach up to 50%.
Another cold acid-fast staining method was reported to be a good
alternative for M. tuberculosis diagnosis, particularly in smaller laborato-
ries and peripheral centers (Gokhale et al., 1990; Madan et al., 1999). Its
shortened dyeing time and the elimination of the need for heating are
two advantageous characteristics of the cold acid-fast staining method.
In this method, if the primary stain is kept for 20 min, the method's
sensitivity is equal to that of the traditional Ziehl–Neelsen method.
However, if the primary stain is kept for only 10 min, the method's sen-
sitivity decreases substantially (Pandey et al., 2009). Heating was also
not needed in our method, and the staining procedure duration was
only about 5 min. Our modified acid-fast staining method is rapid and
sensitive; thus, it is suitable for use in a general tuberculosis laboratory.

Table 1
Comparison of results from our modified acid-fast staining method and the PCR method.

PCR method Our modified acid-fast staining method Total

Positive Negative

Positive 12 3 15
Negative 1 34 35
Total 13 36 50

Statistical parameters of our method compared to PCR method: sensitivity = 80%,
specificity = 97.1%, positive predictive value=92.3%, negative predictive value=94.4%,
and diagnosis efficiency=92%.
No significant differences of the positive rates were found between our method and
PCR method (Corrected χ2 = 0.25, P = 0.6171 > 0.05).
Fig. 3. Positive detection rates for MTB and L-MTB as measured by three methods. Method A,
traditional Ziehl–Neelsen acid-fast staining method; method B, modified IK acid-fast staining
method; and method C, our modified acid-fast staining method. Significant differences
(Pb 0.05) compared with method A indicated by * and △, while # indicates no significant
difference (P>0.05) compared to method B.
132 D. Zhao et al. / Journal of Microbiological Methods 91 (2012) 128–132
Conflict of interest statement
Declaration of interest: the authors report no conflicts of interest.
The authors alone are responsible for the content and writing of the
paper. This paper has not been published elsewhere, and it has not
been submitted simultaneously for publication elsewhere.
Acknowledgment
Our study was supported by the Science and Technology Program of
Luohu District, Shenzhen, Guangdong Province, China (grant 2009014).
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