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Modified detergent Ziehl-Neelsen technique for the staining of Cyclospora

cayetanensis

Article  in  Journal of Clinical Pathology · July 1996

DOI: 10.1136/jcp.49.6.511 · Source: PubMed

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J Clin Pathol 1996;49:511-512 511

Short reports

M4odified detergent Ziehl Neelsen technique for

the staining of Cyclospora cayetanensis

S C Clarke, M McIntyre

Abstract Methods
Cyclospora cayetanensis is a cause of pro- Two separate lots of carbol fuschin solution
longed diarrhoea, mainly in travellers. were prepared, one containing phenol, the
Laboratory diagnosis may be achieved by other containing detergent. The former was
a number of methods such as the staining prepared according to the method of
of faecal smears by the modified Ziehl- Casemore,' whilst the latter was prepared as
Neelsen (ZN) technique. Safer methods follows: 0.2 g carbol fuschin powder was added
using this technique have been described to 20 ml absolute alcohol and mixed well. After
for the staining of acid fast bacilli and the former was completely dissolved, 80 ml
cryptosporidia by replacing the phenol distilled water was added and gently mixed,
content of the carbol fuschin stain with followed by 30 ml Amway LOC Regular deter-
various concentrated detergents. In this gent. Acid-alcohol and malachite green com-
report the technique was modified slightly ponents of the staining technique were also
using a non-concentrated detergent and prepared as previously described by
applied to the staining of oocysts of Casemore.'
C cayetanensis. It was found that oocysts Faecal smears of a sample known to contain
of C cayetanensis do not stain using the oocysts of C cayetanensis and Cryptosporidium
modified detergent ZN method when spp., respectively, were then stained following
compared with similar preparations con- the method of Casemore,' but using the deter-
taining oocysts of Cryptosporidium spp. gent carbol fuschin solution as opposed to the
(7 Clin Pathol 1996;49:51 1-512) phenol carbol fuschin solution. Control stain-
ing was performed in parallel by using smears
Keywords: Cyclospora cayetanensis, Ziehl-Neelsen stain, of the same samples and staining them follow-
detergent. ing the method of Casemore2 using the phenol
carbol fuschin solution. The staining methods
were repeated at least 10 times to ensure that
Cyclospora cayetanensis is an established cause consistent results were obtained.
of prolonged diarrhoea. Cases of infection have
been reported worldwide but may be seen most Results
frequently in individuals who have returned Using the modified detergent ZN method,
from Central and South America or South and oocysts of C cayetanensis completely resisted
South East Asia. Laboratory diagnosis may be staining and appeared as non-refractile cysts
achieved by examining wet mount preparations against a blue-green background. They could
of faeces under direct light microscopy, by often be seen due to the differentiation from
examining wet mount preparations for auto- the background. However, they were also often
fluorescence under ultraviolet epifluorescencel missed due to the lack of staining. Oocysts of
or by staining faecal smears by the modified Cryptosporidium spp. stained pink by this
Ziehl-Neelsen (ZN) technique.2 The latter method.
may be used in some laboratories for the diag- Using the modified phenol ZN method,
Department of nosis of cryptosporidiosis. The same technique oocysts of C cayetanensis stained variably. Some
Microbiology, may be easily applied to screening for C cayet- stained dark red with a mottled appearance,
Wexham Park anensis' even though screening may be easier by some stained pink, whilst others did not stain
Hospital, using ultraviolet epifluorescence.4 all and appeared as non-refractile glassy
Slough, Berkshire
SL2 4HL A safer modified ZN method for the staining spheres against the blue-green background.
of acid-alcohol fast bacilli has been described.5 Oocysts of Cryptosporidium spp. stained dark
Correspondence to:
Mr S C Clarke,
The technique involves the substitution of pink and the staining was less variable.
Department of Microbiology phenol used in the carbol fuschin solution with
and Immunology, a concentrated liquid organic detergent. Phe- Discussion
University of Leicester,
School of Medicine, nol is both cytotoxic and carcinogenic. The The modified detergent ZN stain did not per-
Medical Sciences Building,
PO Box 138,
substitution of phenol for detergent has also form at all well when compared with the results
University Road, Leicester been described for use in the modified ZN seen using the modified phenol ZN. This was
LE1 9HN. method for the staining of cryptosporidia.6 The because oocysts of C cayetanensis did not hold
Accepted for publication technique has not yet been described for the the detergent carbol fuschin stain. A number of
20 February 1996 staining of oocysts of C cayetanensis. detergents have been used for this technique
 Downloaded from jcp.bmj.com on October 20, 2011 - Published by group.bmj.com

512 Clarke, McIntyre

including Amway LOC High Suds and BDH the intensity of staining was not as good as seen
Neutracon. Unfortunately, the former is no in the modified phenol ZN. The auramine-
longer available but LOC Regular, which is a phenol technique is probably more widely used
less concentrated formula, was used at a higher for the staining of cryptosporidia. C cayetanen-
concentration than has been previously de- sis does not stain well by this technique and
scribed to overcome this difference. It is therefore a separate procedure should be
suggested that although the method may be followed for the laboratory diagnosis of this
cheaper and is safer than the modified phenol organism.
ZN due to the absence of phenol, it should not
be used for the modified ZN staining of C cay- This work was completed in part fulfilment of a higher degree at
etanensis. The technique should not therefore the University of Surrey.
be applied to the staining of cryptosporidia
because incidental findings of C cayetanensis 1 Chiodini PL. A 'new' parasite: human infection with
may be missed. Although the presence of Cyclospora cayetanensis. Trans R Soc Trop Med Hyg
1994;88:369-71.
oocysts of C cayetanensis could be detected in 2 Casemore DP. Laboratory methods for diagnosing
the preparations, the technique could not be cryptosporidiosis. J Clin Pathol 1991 ;44:445-51.
3 Clarke SC. Laboratory diagnosis and autofluorescence of
used for screening purposes in a diagnostic Cyclospora. BrJ7 Biomed Sci 1995;52:23 1.
4 Dytrych JK, Cooke RPD. Autofluorescence of Cyclospora.
laboratory because the possibility of error BrJ7 Biomed Sci 1995;52:76.
would be too great. Oocysts of Cryptosporidia 5 Ellis RC, Zabrowarny LA. Safer staining method for acid
spp. have been shown to stain well by others6 fast bacilli. J Clin Pathol 1993;46:559-60.
6 Kelly B. Safer staining method for acid fast bacilli (letter).
and did so satisfactorily in this study. However, JClin Pathol 1994;47:286.

I Clin Pathol 1996;49:512-514

Gelatinous degeneration presenting as a

preleukaemic syndrome
R Arranz, J J Gil-Fernandez, A Acevedo, J F Tomas, A Alegre, J M Fernandez-Rafiada

Abstract Gelatinous degeneration is generally diagnosed

Gelatinous degeneration of marrow is a in bone marrow biopsy specimens by the pres-
rare histological disorder associated with ence of a focal or generalised extracellular
chronic debilitating diseases, such as deposition of a gelatinous material, identified
anorexia nervosa, AIDS and postchemo- as hyaluronic acid, in association with fat atro-
therapy aplasia. Solid tumours have been phy and marrow hypoplasia. This disorder has
associated with this condition but it has been referred as serous atrophy, mucoid
been reported in only two patients with degeneration and starvation marrow.' Gelati-
leukaemia. In these cases leukaemia and nous degeneration has been classically ob-
gelatinous degeneration were diagnosed served in association with chronic debilitating
simultaneously. In the case reported here, disorders, such as anorexia nervosa, starvation,
Department of
a 48 year old man, gelatinous degeneration malignancy, chronic infections, systemic lupus
Hematology, was the only histological finding observed erythematosus, and myxoedema.'-5 Recently, it
Hospital Universitario more than two years before the diagnosis has been widely reported in patients with
de la Princesa, of acute myelogenous leukaemia with AIDS and a variant form of the classical
Madrid, Spain
R Arranz monosomy 7. The significance of hyalu- degeneration has been reported after the
J J Gil-Fernandez ronic acid deposition remains uncertain. administration of chemotherapy.8
J F Tomas
A Alegre
Two hypotheses have been put forward: (1) In the case reported herein, gelatinous
J M Fernandez-Ran'ada
that gelatinous degeneration occurs dur- degeneration was diagnosed in a previously
ing tissue repair; and (2) that gelatinous healthy man, with no evidence of an underlying
Department of degeneration inhibits haemopoiesis by disorder. To our knowledge, this is the first
Pathology altering the microenvironment of the reported case in which gelatinous degeneration
A Acevedo
bone marrow. In the case reported here, preceded the diagnosis of acute myelogenous
Correspondence to: the presence of monosomy 7 suggests that leukaemia (AML).
Dr Reyes Arranz,
Servicio de Hematologia y myelodysplasia was the underlying disor-
Hemoterapia, der which finally evolved into acute leu-
Hospital Universitario de la kaemia. Case report
Princesa,
Diego de Le6n, 62, 28006 ( Clin Pathol 1996;49:512-514) A 48 year old healthy, well nourished, white
Madrid, Spain. man was admitted to hospital in August 1992
Accepted for publication Keywords: gelatinous degeneration, acute myelogenous because of fever and pancytopenia. He worked
12 December 1995 leukaemia, monosomy 7. as a driver for a chemical company, smoked
 Downloaded from jcp.bmj.com on October 20, 2011 - Published by group.bmj.com

Modified detergent Ziehl-Neelsen technique

for the staining of Cyclospora cayetanensis.
S C Clarke and M McIntyre

J Clin Pathol 1996 49: 511-512

doi: 10.1136/jcp.49.6.511

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