Journal of Microbiological Methods 91 (2012) 128–132

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Journal of Microbiological Methods

journalhomepage: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

article info abstract

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

Corresponding author at: Department of Laboratory, Luohu Chronic Disease Prevention and 2.1. Patients
Cure Hospital, Shenzhen 518023, China. Tel.: +86 755 8245 1811; fax: +86 755 8205 6724.
E-mail addresses: Olivia_dan@126.com (D. Zhao), sysu_microbiology@126.com (X.-
M. Yang), chexiaoyan@126.com (X.-Y. Che). Four hundred and sixty-eight patients with confirmed tuberculo-sis from
Luohu Chronic Disease Prevention and Cure Hospital were included in our
0167-7012/$ – see front matter © 2012 Elsevier B.V. All rights reserved. study.
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 ð true 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 b0.05 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 (Pb0.05)
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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