Validation of sterilization of Lucilia cuprina larvae using raw honey through scanning electron microscope

Background: In the application of Maggot Debridement Therapy (MDT), sterilized maggots (Lucilia cuprina) by standard chemicals were used. Recent potential demands for using a procedure with allnatural products, raw honey has been proposed as a natural sterilization technique to replace the chemical products. Methods: Lucilia cuprina collected from a laboratory colony were divided into treated group (sterilized) and control group (non-sterilized) to undergo sterility testing with honey dilution. Scanning electron microscope (SEM) was used to examine and ascertain the sterility of honey dilution. SEM provided direct observation of microorganism’s presence on external surface L. cuprina after sterilization by disinfectants of raw honey (tualang honey). Results: Treated group showed the absence of microorganism on the surface body segment of the larvae. It refl ected in different magnifi cations in which treated maggots appeared to be smooth and clear of any external contaminants. The untreated maggots exhibited clusters of microorganisms on the external body surface. Conclusion: This study confi rmed the complete sterilization of maggots using raw honey as the antibacterial agent using SEM due to its disinfection effi cacy in destroying microorganisms in L.cuprina. Research Article Validation of sterilization of Lucilia cuprina larvae using raw honey through scanning electron microscope Nur Afrina MH1*, Santhana Raj L1, Nur Fateha R1, Wan Nur Alia WZ2, Kamesh R3 and Nazni WA2 1Electron Microscopy Unit, Institute for Medical Research, Ministry of Health Malaysia, Jalan Pahang, 50588 Kuala Lumpur, Malaysia 2Entomology Unit, Institute for Medical Research, Ministry of Health Malaysia, Jalan Pahang, 50588 Kuala Lumpur, Malaysia 3Environmental Health Research Centre, Institute for Medical Research, Ministry of Health Malaysia, Jalan Pahang, 50588 Kuala Lumpur, Malaysia Received: 16 October, 2019 Accepted: 30 October, 2019 Published: 31 October, 2019 *Corresponding author: Nur Afrina MH, Electron Microscopy Unit, Institute for Medical Research, Ministry of Health Malaysia, Jalan Pahang, 50588 Kuala Lumpur, Malaysia, E-mail:


Introduction
Maggot Debridement Therapy (MDT) became an emerging trend as a natural remedy that can heal wounds. For the application of MDT, sterilized blow fl y (Lucilia cuprina), commonly called maggots, were used. The MDT product used sodium hypochlorite and formaldehyde as the main disinfectants of the maggots. This had been validated by using a Scanning Electron Microscopic (SEM) that identifi es the absence of bacteria on maggots, in which this process has been approved by the Ministry of Health of Malaysia as a safe procedure to use [1].
Due to increasing demand by potential clients who want to use a procedure with all natural products, raw honey has been proposed as a natural sterilization technique to replace the chemical products. Raw honey was discovered as an effective therapeutic agent because it contains potent antimicrobial action against a broad spectrum of bacteria and fungi [2]. Jenkins (2012) noted that all types of honey have high hydrogen peroxide, sugar content and acidity, which prevents microbial growth [3]. Glucose and fructose oxidization will also produce A hydrogen peroxide, which can inhibit and destroy bacteria cell walls. When honey is diluted with water, the enzymes create a small amount of hydrogen peroxide that gives an exothermic effect of being bactericidal [4].
Although it has not been proven that honey actually sterilized maggots, this study has the potential to be explored further. This helps us in exploring extensively the usage of raw honey as an antibacterial agent. This fi nding could provide avenue for further research on honey, in sterilizing the maggots as chemical usage can be reduced. This study also evaluated the process of sterilization of maggots using raw honey, validated with an SEM.

Raw honey
Pure raw honey (tualang honey) was obtained from Federal Agriculture Marketing Authority (FAMA), Malaysia, which was collected from Koompassia excelsa (tualang tree). For the sterilization process, the honey was diluted with distilled water (1:10). All the assays were performed in laminar fl ow. Preparation for the sterility of maggots L. cuprina maggots (3 rd instar larvae) were removed from the cow liver and divided into treated and control groups.
The raw honey was diluted with distilled water prior to the sterilization process. The dilution prepared is one part of raw honey in nine parts of sterile distilled water in the ratio of 1:10. The treated group was surface sterilized with honey dilution by stirring for 5 minutes then washed with sterile distilled water. This process was repeated three times.
The untreated group was only washed in sterile distilled water.

Preparation of maggots for SEM
Sixty maggots were examined with 30 in each group under SEM in three magnifi cations; x300, x1500, x6500. The treated and untreated maggots went through SEM processing procedure using increasing concentrations of ethanol 70%, 90% and 100% (3 times) in 5 minutes respectively for dehydration.
Subsequently the maggot samples were dried by using critical point dryer (CPD). Therefore, the ethanol is replaced by CO 2 fl uid under 1072 psi pressure (10°C). After complete exchange of fl uids, the maggot samples were heated up to 40°C. By heating up, the CO 2 fl uid would be phased into CO 2 gas. Then the CO 2 gas were gently released from the CPD device until atmospheric pressure is obtained. The dried material was mounted on holders or stubs, sputter coated with a thin layer of conductive material (42nm gold thickness) and then ready to be viewed by SEM (Philips XL-30 ESEM, IMR, Malaysia). The fl ow chart of the SEM processing is shown in Figure 1.

Results
The treated maggot's group showed absence of microorganisms on the surface body segment of the larvae On the other hand, the untreated maggots exhibited clusters of microorganisms on the external surface with three different SEM magnifi cations ( Figure 3). The microorganism species were not identifi ed or differentiated, but however, by observing the magnifi cations of the electron microscopic images, we noticed that they were more cocci-like and less bacilli-like.
This was because Cocci bacteria were more commensal to the environment compared to Bacillus bacteria.

Discussion
Treated maggots with raw honey (tualang honey) showed a sign of complete sterilization as judged by absence of bacteria or any other microorganisms. However, untreated maggots showed presence of bacteria and other microorganisms on the external surface of maggot samples. The bacteria and microorganisms found were categorized as contaminants. Although we never carry out blood agar culture test to emphasize the complete sterilization test, observation done by SEM showed clear surface on the maggots as sterilization can be accomplished by diluted raw honey.
This preliminary study determined the effi cacy of raw honey to sterilize maggots. From previous studies, several species of bacteria isolation had been demonstrated from L. cuprina such as pathogenic E. coli, which causes diarrhea [5]; and P. mirabilis, which causes urinary tract infection and systemic infl ammatory response syndrome [6]. It is particularly interesting that we also detected cocci-like bacteria that was often regarded as contaminants.
This study provided SEM as a validation tool to confi rm the sterility of the maggots produced after sterilization before MDT application. Our results indicated that sterility of maggots  Sterilization process used in MDT is a rigorous process that results in sterility but produces a high number of eggs after the sterilization process [7]. However, in this study only 3rd instar larvae were used as a proxy to determine the effi cacy of raw honey in sterilizing the larvae surfaces. Baer (1931) reported that it was necessary to use sterile maggots on patients [8]. In his early experiment, non-sterile maggots were used as MDT debriding agent; however, patients subsequently developed secondary infection such as tetanus.
In addition to Baer (1931), Nuesh et al., (2002) also concluded that it was essential to use only sterile maggots on patients.
Recent studies showed that the best sterilization method is to sterilize the eggs as compared to sterilization of maggots, and this observation was in line with Baer's fi nding [8]. In this study, we used 3 rd instar larvae as the proxy instead of eggs to get preliminary data in short period of time to ensure this study works effectively that could lead to further research.
For this study, raw tualang honey was selected as the disinfectant to sterilize L. cuprina surface without affecting its larval survival. The sterilization process was successful which was confi rmed through SEM evaluation. In raw honey, the properties that contributed to antibacterial activity were hydrogen peroxide (H 2 O 2 ), high osmolarity, and presence of phytochemical components (non-peroxide) [9]. multiplication of bacteria will not happen. Directly, the bacteria will be killed and do not cause primary and secondary infections [10]. This proves that raw honey has the antibacterial effect and confi rms our obtained results.
As mentioned above, H 2 O 2 will puncture holes on the outer membrane of the bacteria which will cause differential osmotic pressure between the internal and external surface of the bacteria. Whereby, the external osmotic pressure will cause a blown-up effect that will rupture the outer membrane of the bacteria [11]. By the blow-up effect, antibacterial activity of raw honey will be more compressive.