Los manuales escolares de ELE para las aulas en Rumanía

Nascimento (1992) used Salmonella as the target microbe in his experiment. In his

study the eggs were just rinsed in distilled water, as Salmonella can be easily isolated and distinguished as it differs from the normal microflora of chicken eggs they did not need to be sterile. However, in my study the bacteria being tested were ones that had been isolated from wild eggs. Therefore, in order to determine that the bacteria placed on the shell was the same bacteria that grew through the shell, and not for example a microbe already within the shell, the egg fragments needed to be sterilised prior to use. Effectively, any method used to clean eggshells could potentially compromise the shell structure and increase the penetrability by the bacteria being tested. For example harsh, abrasive cleaning has been shown to remove the cuticle layer (Sparks, 1994; Rideout, 2012). The cleaning method chosen in this study ideally should not overly compromise the shell structure.

6.3.6.2 Method

Several eggshell cleaning methods were initially trialled using chicken eggshell fragments and then NIBK eggshell fragments (see table 6.3 and table 6.4). I used two controls, unclean shells and shells washed using distilled water, I expected both of them to allow the growth of bacteria. Ethanol, ultraviolet light and a combination of both were used, as these methods are often used to disinfect equipment and surfaces (Macpherson, 2004). Autoclaving and pressure cooking were also trialled because these methods are known to be able to sterilise medical equipment (Rutala and Weber, 1999). ONE uses Incusan™ to clean the NIBK eggs they receive (Bassett, 2012), This was not available to me in this study however, chemical cleaning, such as ethanol and Incusan™ may only disinfect, and not sterilise, the shell fragment (A. Midwinter pers. comm.).

Figure 6.6: Photo of a North Island Brown Kiwi eggshell fragment being checked for contamination after a sterilising trial. The shell fragment has been taken off the agar, the black circle indicates where to swab under shell to check for contamination.

Once the shells had been cleaned by the designated method, they were set into agar using the methods described in trials one and five and incubated at 36oC for 48 hours. The shells were visually checked after 24 and 48 hours incubation. However, visually observing growth on the surface of the agar was not enough to determine penetration, as microbial growth is not always visible on the surface (see trial eight). The agar under the shell fragments needed to be swabbed and re-plated to be certain no growth occurred (see figure 6.6). Therefore, after 48 hours incubation each shell fragment was

removed from the agar and the surface of the agar under the shell was swabbed (see figure 6.6) and plated onto a new blood agar plate. This re-sub was incubated for further 48 hours at 36oC to confirm bacterial growth.

Table 6.3: List and description of the eight methods trialled in this study to attempt to clean eggshell fragments.

Cleaning method trialled Description of method

No cleaning No cleaning attempt was made

Sterile water 500ml of water was squirted onto both sides of the shell fragment using a sterile wash bottle

Ethanol 500ml of 80% ethanol was squirted onto both sides of the shell fragment using a sterile wash bottle

Ultra-violet light (UV) Shell fragment was placed in a petri dish and held under UV light for 15 minutes

Ethanol and Ultra-violet light (UV)

500ml of 80% ethanol was squirted onto both sides of the shell fragment using a sterile wash bottle. The shell fragment was then placed in a petri dish and held under UV light for 15 minutes

Autoclave (dry run) Shell fragment was sealed in an autoclave sterilisation bag and autoclaved in a Getinge steam steriliser (Getinge Group, Auckland, New Zealand) for 20 minutes at 121oC

Autoclave (fragments set in agar)

The shell fragment was set in agar in a petri dish, this was then autoclaved in a Getinge steam steriliser for 20 minutes at 121oC

Pressure cooker Shell fragment was placed into a beaker then put in an Model 925 All American Pressure Cooker (All American Cooker, Hillsville, Virginia, USA), for 20 minutes at 15 psi and 120oC

To determine if contamination was occurring due to handling error, swabs were also taken from two of the NIBK shell fragments directly after coming out of the pressure cooker trial.

6.3.6.3 Results

Initial trials using un-cleaned eggs resulted in growth on the agar for both chicken and NIBK eggshells as expected. Sterile water failed to clean chicken eggshells and bacterial growth was seen after incubation; for this reason, I did not use cleaning with distilled

water on NIBK eggs. Ethanol, ultraviolet light and a combination of both resulted in a reduction in bacterial growth on the chicken eggs, but there was still heavy growth on the NIBK shell fragment. When the chicken eggshells were place straight into the autoclave without agar, the shell membranes visibly shrived and pulled away from the shell. When the NIBK eggshell was placed into the autoclave without agar, the membranes did not visibly dry out but heavy growth still occurred when the agar under the shell was swabbed after 48 hours. The chicken eggs that were set in agar before being placed in the autoclave did not dry out, but the agar bubbled up vigorously over and around the shell fragment. This bubbling could result in the agar being pushed through the pores, which would compromise the results of this study as it could increase the penetration of bacteria, thus this method was abandoned. Instead, a pressure cooker was used, as in my experience this creates a wetter environment than the autoclave. The pressure cooker successfully sterilised the chicken eggs however NIBK eggs still showed growth after 48 hours (see figure 6.7). Both NIBK eggshell fragments that were swabbed directly after the pressure cooker treatment showed heavy growth, indicating that the contamination was present before cleaning took place.

Figure 6.7: Comparison of the bacterial growth present after attempts to sterilise North Island Brown Kiwi eggshells. Fragments were pressure cooked at 15 psi for 20 minutes, then set into agar and incubated at 36oC for 24 hours and 48 hours.

Due to restraints around sample size, time and budget, attempts to sterilise NIBK eggs had to be halted at this stage. Identification of the bacteria that grew after the cleaning trials was out of the budget of this study, but samples have been kept in storage for future work (see chapter seven, section 7.3).

Table 6.4: Comparison of the sterilising treatments used on North Island Brown Kiwi (NIBK) and chicken eggshell fragments. Fragments were treated, set in agar and incubated. Observations of bacterial growth were noted visually after 24 hours, then checked by a re-sub of the agar under the shell after 48 hours. - = not tested.

Treatment

Chicken eggshells NIBK eggshells

24 hour (visual check) 48 hour (under shell agar swab) 24 hour (visual check) 48 hour (under shell agar swab)

No cleaning Growth Growth Heavy growth Heavy growth

Sterile water Growth Growth - -

Ethanol Growth

Less than control

Growth Heavy growth Heavy growth

Ultra-violet light Growth

Less than control

Growth Heavy growth Heavy growth

Ethanol and UV light Growth

Less than control

Growth Heavy growth Heavy growth

Autoclave (dry run) No growth

Membranes dried out completely

No growth No growth Heavy growth

Autoclave (fragments set in

agar)

No growth Agar bubbled over

shell surface

No growth - -