Tropical Biomedicine 28(2): 325–332 (2011)

Scanning electron microscopic evaluation of the successfulsterilization of Lucilia cuprina (Wiedemann) utilized inmaggot debridement therapy (mdt)
Yeong, Y.S.1,3, Nazni, W.A.1*, Santana, R.L.2, Mohd Noor, I.1, Lee, H.L.1 and Mohd Sofian, A.3
1 Medical Entomology Unit, Infectious Diseases Research Centre (IDRC), Institute for Medical Research,Jalan Pahang, 50588 Kuala Lumpur, Malaysia
2 Electron Microscopy Unit, Institute for Medical Research, 50588 Kuala Lumpur, Malaysia
3 Institute of Biological Science, Faculty of Science, University of Malaya, 50588 Kuala Lumpur* Corresponding author email: nazni@imr.gov.myReceived 1 November 2010; received in revised form 19 February 2011; accepted 28 February 2011

Abstract. In Malaysia, maggot debridement therapy (MDT) utilizes maggots of Lucilia
cuprina (Wiedemann) to debride necrotic tissue from wound surface, reduce bacterial infection
and therefore, enhance wound healing process. To evaluate the sterility of the sterile maggots
produced after sterilization process before delivering onto patient wounds. Sterility of sterilemaggots is crucial in ensuring the safe usage of MDT and patient’s health. Eggs of L. cuprina
collected from a laboratory colony were divided into treated group (sterilized) and control
group (non-sterilized). Treated group underwent sterilization while eggs from control groupwere allowed to hatch without sterilization. Sodium hypochlorite and formaldehyde were themain disinfectants used in this sterilization process. Scanning electron microscope (SEM)
was used to examine and ascertain the sterility of sterile maggots. SEM results showed thatall sterilized L. cuprina eggs and maggots achieved sterility and all were cleared frombacterial contamination. In contrast, all non-sterilized eggs and maggots were found to becolonized by microorganisms. Sterilization method employed to sterilize eggs and maggots
used in Malaysia MDT was proven successful and MDT is safe to be used as wound management
tools.

2004). In Malaysia, the tropical bronze
blowfly, Lucilia cuprina is utilized. MDT waslong established and widely used in the USA
since 1930. Malaysia is the first to conduct
MDT research (Mohd Masri et al. , 2005) inSoutheast Asia region and this treatment isgradually being adopted by local hospitals
and private clinics to benefit patients
suffering from non-healing wounds (Massari,et al., 2008).
In Malaysia, the first MDT clinical trial
dated back to 2003 with 12 patients sufferingfrom diabetic ulcers. Ten out of 12 patients
healed satisfactory after MDT treatment(Rajoo, 2004). Malaysia’s first MDT casecontrol clinical study was conducted in 2008
and MDT was proven to be as efficient asconventional treatment (Rajoo, 2004; Aaron

INTRODUCTION
Maggot debridement therapy (MDT) is a formof therapeutic wound treatment utilizing
maggots of certain blowfly species to remove
non-vitalized tissue, pus, slough andmetabolic wastes on the wound (Hebda & Lo,2001). Maggot debridement therapy hastenshealing time and therefore decreases
hospitalization duration. Wound healed by
MDT appeared to have smaller scar becauseof the rapid proliferation of healthy
granulation tissue (Sherman & Pechter,1988).
In temperate countries such as UnitedStates of America, United Kingdom andEurope, Lucilia sericata is being employed
as a debriding agent in MDT (Geary & Russel,325

et al., 2009). Thus far, no complication anddetrimental effects are reported fromphysicians, nurses and patients who employ
or utilizing MDT.
Sterilization is the process of sterilizingthat completely destroys or removes all living
microorganisms (Rutala & Weber, 1999).
The Council on Pharmacy and Chemistryof U.S in 1936 noted that sterilizationmeans the absence or total destruction of
all microorganisms (American MedicalAssociation, 1936). Sterilization of the eggsof L.cuprina for use in MDT is vital and is a
pre-requisite before using them for MDT
application. This step is crucial to ensure thesafe usage of sterile maggots on patients’
wounds and their health.
The present paper reports the firstattempt to utilize scanning electron
microscope (SEM) to evaluate the sterilityof sterile maggots produced and effectivenessof the sterilization process used in maggot
debridement therapy utilizing the Malaysian
blowfly, L. cuprina.

that hatched from treated eggs and control
eggs were fixed with 2.5% glutaraldehyde
in 0.1 normal phosphate buffer solution
and were immediately sent to Electron
Microscopy Unit of Institute for Medical
Research for SEM evaluation. They werethen processed by dehydrating using a graded
series of ethanol solutions, coated with gold
and viewed using SEM.

RESULTS
The scanning electron microscope (SEM)
results on both treated (sterilized) and control(non-sterilize) eggs showed clear andsignificant differences in term of sterility.
Significant differences between nonsterilized egg and sterilized egg wereobserved under SEM. Contaminants wereseen adhering to the external surface of nonsterilized egg (Figure 1), whereas sterilized
egg appeared to be smooth and clean andclear of any external contaminants (Figure2). Vastly different SEM view of the nonsterilized and sterilized egg surfaces wasshown on Figures 3 and 4. Non-sterilized eggs
exhibited clusters of microbial flora on its
external surface (Figure 3), while externalsurface of sterilized egg was clean under thesame magnification (Figure 4). Figure 5
clearly showed colonization of microbial
flora on the outer surface of non-sterilizedegg, while Figure 6 indicated the structure ofplastron in an egg at the magnification of10748X. The plastron is a widely spaced
meshwork of the eggshell that allows air totrap within it for ensuring respiratoryexchange during period of immersion(Bursell, 1970). No bacterial contaminants
were trapped within this fine mesh of plastron(Figure 6).
After 24 hours of incubation period, eggs
were hatched into first instar maggots.
Maggots that hatched from sterilized andnon-sterilized eggs were fixed withglutaraldehyde and sent to Electron
Microscopic Unit of IMR for evaluation.
Figure 7, clearly showed the colonization ofbacteria on the anterior head of non-sterilized
L. cuprina maggot, whereas Figure 8
showed clean and clear anterior head ofMATERIALS AND METHODS
Flies
Fly colony of L. cuprina used in this study
was bred and maintained in the insectariumof Institute for Medical Research (IMR),Kuala Lumpur. They were kept at 27-29ºC
with relative humidity of about 70%. They
were fed with water and castor sugar as
routine supplement. Pieces of cow liver weregiven to flies as protein source to induce
oviposition as well as an oviposition medium.
Lucilia cuprina maggots
Eggs were removed from the cow liver anddivided into treated and control group. The
treated group was surface sterilized with
0.05% sodium hypochlorite followed by
5% formaldehyde and then washed withsterile distilled water. The eggs were thentransferred onto agar plates and incubated at28ºC for 24 hours (Wolff & Hasson, 2005). The
control group was allowed to hatch without
undergoing sterilization and were transferred
onto sheep blood agar and kept separately
inside an incubator. After 24 hours, maggots
326

Figure 1. Contaminated outer surface of nonsterilized L. cuprina egg. M: 196X

Figure 2. Clean and clear outer surface ofsterilized L. cuprina egg. M: 204X

Figure 3. Clumps of microorganisms on the outersurface of non-sterilized L. cuprina egg. M: 2687X

Figure 4. Clear and smooth outer surface ofsterilized L. cuprina egg. M: 2687X

Figure 5. Clusters of cocci-liked bacteria on theouter surface of non-sterilized L. cuprina egg.
M: 10748X

Figure 6. Plastrons of L. cuprina sterilized egg
can be observed free of microorganism. M:10478X

327

Figure 7. Colonization of bacteria on theanterior head of non-sterilized L. cuprina
maggot. M: 2687X

Figure 8. Absence of microbial flora on theanterior head of sterilized L. cuprina maggot.
M: 1344X

sterilized L. cuprina maggot. Figures 9, 11,and 13 indicated bacillus-like bacteriacolonizing the hair-like structure on the bodyof maggot and body segment of non-sterilized
L. cuprina maggot, however, Figures 10, 12and 14 showed absence of the bacillus-likebacterial colony on similar body section ofL. cuprina maggot. In Figures 10, 12 and 14,
spots that appeared adhering to the bodysegment were possibly food debris and lysedmaterials.

disinfecting procedures used in MDT;
utilizing L. sericata instead of P. terranovae
and application of MDT was reported safe
since then. Baer (1931) also reported that itwas necessary to use sterile maggots onpatients. In his early experiment, non-sterilemaggots were used as MDT debriding agent;
however, patients subsequently developed
secondary infection such as tetanus (Baer,1931).
In addition to Baer (1931) and Nuesh etal. (2002) also concluded that it wasindispensible to use only sterile maggots onpatients (Baer 1931; Nuesch, et al., 2002). In
our studies, the best sterilization method isto sterilize the eggs as compared tosterilization of maggots and this observation
was in line with Baer’s finding (Baer, 1931).
He reported that the external surface ofmaggots could be sterilized, but not thecontents of the gut and intestines of maggots.
Therefore, the maggots continued to be septic
as they constantly secreted gut contents into
the surrounding environment.
Lucilia cuprina eggs are laid in cluster
which must be separated into single egg
before sterilization process to ensure
disinfectants used can sterilize the entire
external surface of the egg (Baer, 1931).
Generally, contamination can be easily
detected after sterilization process as
sterilized eggs will be deposited onto sterile
agar plate and incubated for 24 hours
before the maggots hatch from the eggs.
Contamination on the agar plates will be

DISCUSSION
The present study showed that scanningelectron microscope is also a promising
method to confirm the sterility of thesterilized eggs and sterile maggots produced
after sterilization before MDT application.
Our results indicated that sterility of sterile
eggs and maggots was successfully achieved
after sterilization.
Sterilization process used in MDT must
be a stringent process resulting in totalsterility yet yielding high number of viable
eggs after sterilization process (Nuesch et al.,2002). This is to ensure large number of sterilemaggots can be produced at any one time tomaximize the cost -effectiveness of MDT.
The same author (Nuesch, et al., 2002)
reported an unusual clustering of bloodstream
infection of five of their patients after using
contaminated larvae of Protophormia
terranovae. Later on, they improved the328

Figure 9. Bacillus-liked bacteria colonized thehair-liked structure of non-sterilized L. cuprina
maggot. M: 10749X

Figure 10. Clean surface observed on the bodyof sterilized L. cuprina maggot. M: 10748X

Figure 11. Bacillus-liked bacteria colonized thesegment of non-sterilized L. cuprina maggot.
M: 2687X

Figure 12. No colonization of bacteria foundon the body segments of sterilized L. cuprina
maggot. M: 2687X

Figure 13. Magnified bacillus-like bacteriacolonized on the body of non-sterilized L.
cuprina maggot. M: 10748X

Figure 14. Only debris and possible lysedmaterials on the body sterilized L. cuprina
maggot. M: 10748X

329

indicated by growth of microorganisms
which could be easily detected (Sherman &Wyle 1996).
Simmons (1934) suggested that few
factors needed to be considered before
choosing a disinfectant for sterilization
process for MDT (Simmons, 1934). There areagglutination and floating caused by effectof disinfectants and sterilization, time
consumed for production of sterile maggots
and the age of the eggs used at the time ofsterilization process.
Entomologists use sodium hypochlorite
to surface sterilize the insect eggs andequipments used in limited or mass rearingof insect (Soo Hoo et al., 1971). Linville &Wells (2002) reported that using 20% bleach
to wash or surface sterilize blowfly’s maggots
appeared to be an effective method tosignificantly reduce the risk of external
contamination of maggots.
Sodium hypochlorite and formaldehyde
are antimicrobial agents (Neely, 1963; Estrela& Ribeiro, 2003). Sodium hypochlorite is apotent disinfectant in which the activity
occurs when hypochlorous acid is formedwith the release of chlorine gas (Penick &Osetek, 1970). Sodium hypochlorite has abroad spectrum of antimicrobial activity
and it exhibits robust and rapid activity
against both vegetative bacteria andMycobacterium tuberculosis even at relative
low concentrations (50 ppm). Sodium
hypochlorite inactivated many viruses at 200
ppm of concentrations, such as the Human
Immunodeficiency Virus (HIV) which issusceptible to sodium hypochlorite at 50 ppm
and Hepatitis B Virus (HBV) which isinactivated at 500 ppm (Sharbaugh, 1998).
Formaldehyde (methanal, CH 2 O) isbactericidal, sporicidal, and virucidal. It isan extremely reactive chemical with its wellknown rapid reaction with protein, DNA andRNA in vitro. It has been suggested thatformaldehyde acts as a mutagenic agent andas an alkylating agent by reaction withcarboxyl, sulfhdryl, hydroxyl groups andnucleic acid. It also alters Hepatitis B surfaceantigen (HBsAg) and Hepatitis C core antigen(HBcAg) of HBV (McDonnell & Russell,1999). Formaldehyde exerts sporostatic andsporocidal effects on Bacillus subtilis spores(Trujillo & David, 1972).
We found out that the combination ofsodium hypochlorite and formaldehyde
used in our sterilization method prevent
agglutination and thus reducing egg loss
during the sterile maggots production
process. This combination too achieves goodsterility and high eggs viability (85%-90%)
after stringent sterilization process (Yeong,Unpublished data). Our sterilization method
is similar to Simmon’s sterilization process,however, with some modifications. In his
original sterilization method, he used 1%formalin (formaldehyde) with 1% and 5% of
sodium hypochlorite. Each combination wasexposed to 5 and 10 minutes of sterilization.
All combinations used in his study achieved
100% sterility (Simmons, 1934).
Our study is the first description of
successful sterilization of blowfly eggs
confirmed by SEM. We found that thesterilization method employed in sterilizing
eggs of L. cuprina was effective and safe to
be used therapeutically. In addition, ScanningElectron Microscope (SEM) provided us
exceptionally clear and remarkableevidence of the sterility of eggs of L. cuprina.
Acknowledgments. We thank the Director
General of Health, Malaysia, and the Director,Institute for Medical Research, forpermission to publish this paper. This study
was supported by a grant (No. JPP-IMR-06034) from the National Institutes of Health,
Ministry of Health, Malaysia and theMalaysian Technology Development
Corporation Grant for Maggot Therapy(Grant No. 86333-5083). Thanks are also dueto the staff of Medical Entomology Unit, IMR
for their assistance. The authors declare noconflict of interest in this study.

330

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