P. larvae Anaerobic Growth

Goal: Will growth P. larvae on MYPGP agar at 37C for  7 days under two different conditions - anaerobic vs aerobic. A BBL GasPak Anaerobic system (the container) was utilized for the anaerobic treatment. A picture of the anaerobic system is seen below:

Vaseline was applied to cover rim of the container in order to ensure a tight seal when closed. 

 100 uL of Spore Stock B was plated onto four MYPGP agar plates. Two of the plates were placed in the anaerobic chamber, and two were not. A anaerobic gas pack was placed inside the anaerobic chamber (after adding 10 mL of ddH2O to the catalyst) along with two anaerobic indicator strips. The strips turn blue/green in the presence of oxygen, but will turn white in its absence. A picture of the completed complex is seen below:

Anaerobic chamber and two other P. larvae plates were placed in the 37C 5%CO2 incubator.

//EWW

The Waxworm & the P. larvae

From 8-17-14.

There are still a few wax moths that were incubated at 30C that are still alive and moving around. I am unable to see any eggs on the wax paper, so did not transfer any eggs to Diet 3. This is surprising that the wax moths at 30C are surviving so much longer than those at 35C, but did not lay any eggs yet.

Checked the single wax worm that was placed the petri plate. It has definitely increased in size to over half an inch. It appears to have eaten the present BAD (or it dried up) and begun to secrete the webby substances (seen in picture below). An additional 500 uL of BAD was put into the petri plate (seen in picture below). The progress of this wax worm will continue to be monitored.

Dr. Fisher retrieved another retrieved another container of wax worms from a pet store, this time from Timberline viat-bugs (Link). There were 50 wax worms in the container and 17 were removed as they were dead. The remaining wax worms were placed into two 500 mL containers evenly and incubated at 35C. Picture of the two containers is seen below:

//EWW

Probiotic & Antioxidant LC50

From 8-16-14.

Discovered that I had miscounted the number of honey bee larva for this experiment! I originally thought I had 12 total, but in reality I only had 10. Not a big problem, and it certainly didn't effect how the larva were handled during this experiment. The survival percentages just need to be recalculated.
The last larva has died here on day 10 (10/10 larva dead). This marks the last day of this preliminary experiment. With the new and correct number larva in this study here is the breakdown of results:

n=10	Died	% Survival
Day 1	0	100.0%
Day 2	0	100.0%
Day 3	1	90.0%
Day 4	0	90.0%
Day 5	1	80.0%
Day 6	1	70.0%
Day 7	0	70.0%
Day 8	4	30.0%
Day 9	2	10.0%
Day 10	1	0.0%

Quick graph of data:

Compare to P. larvae LC50 Study. There was a reduced mortality rate compared to the P. larvae LC50 study during the early instars, however the mortality by day 9 (end of LC50 study) was similar. Overall, it doesn't appear that there was any effect on the survival of honey bee larva when fed 1000 CFU/mL P. larvae spores with or without the addition of 1000 Cells/mL of yeast. It could be that the yeast had a delaying effect on the P. larvae as the % mortality seen in this study was reduced during the first 6 days compared to the LC50 study. This study would need to be repeated on a larger scale to make a more accurate evaluation of the effect of yeast on P. larvae's ability to cause disease in honey bee larva. 

//EWW

The Waxworm & the P. larvae

From 8-16-14.

No other wax worms have hatched from the BAD plate, nor are any seen in the rearing containers with Diet 3 at this point. The single wax worm that recently hatched from the 10 uL BAD treatment group appears to be doing well and is very lively. It periodically walks through the thin lawn of 1 mL BAD that was placed in its petri dish. It also appears to be larger than yesterday.

Attempted to autoclave one of the 500 mL containers that had been using as a wax moth mating chamber today as well. About 100 mL of H2O was placed into the container and the opening of the container was covered with aluminum foil. Autoclaved at 121C for 20 minutes (liquid cycle on old autoclave in media kitchen). Here is what it looked like after (on the left) compared to an un-autoclaved container (on the right):

//EWW

Probiotic & Antioxidant LC50

From 8-15-14.

Two additional honey bee larva have died today. This brings the total dead to 9 out of 12 (75%) here on day 9. One of those that have recently died displayed the same mysterious white dots on the surface of its body. The survivors do not appear to be in good health (slow to move and still haven't eaten their diets). Some of the larva are seen in the picture below in the 24 well plate.

I will continue to monitor survival.

//EWW

The Waxworm & the P. larvae

From 8-15-14.

Found one other wax worm that hatched from the BAD plate. This one was from the 10 uL BAD treatment group. The wax worm was carefully removed from the 24 well plate and transferred to a petri dish using a sterile inoculation needle. The wax worm seemed reluctant to let go of the needle go onto the polystyrene petri plate. Perhaps the polystyrene is playing a role in the reduced survival rate on the 24 well plate? A volume of 1000 uL of BAD was added to the side of the petri plate and spread out in a thin lawn covering 1/3 of the entire surface. I was certain to cover the edges of the petri dish as the wax worms (and bee larva for that matter) appear to be drawn to those locations. Perhaps this has to do this the increased surface area making them feel more secure, or that during their wandering they simply ran into the edge and don't know where else to go. A picture of the lone wax worm is shown below, it is larger (longer) than the one previously mentioned that is now dead. This one is about 1 cm in length.

Wax worm in petri dish

The petri plate was sealed with parafilm and incubated in an anaerobic chamber at 35C in the Fisher Lab. I will continue to monitor its progress.

No change in the rearing chambers containing Diet 3.

//EWW

P. larvae germination in BAD medium assay

From 7-22-14.

On August 8th, 2014: repeated germination study using Spore Stocks D and E. Three treatment groups were used as before (BHI, H2O, and BAD). Procedure was followed as previously outlined. Briefly, spore stocks were diluted 1:10 and combined with treatment media in a 1:5 dilution. After a 2 hour incubation at 35C 225 rpm blot plate method was performed to quantify CFU/mL. Additionally, a portion was heated to 65C for 15 minutes and the blot plate method was performed again. MYPGP agar plates were incubated at 37C 5%CO2 for 7 days and colony counts were performed today.

Determining CFU/mL from colony count formula/equation:

[# colonies counted / volume pipetted onto plate in uL] * [ 1 / serial dilution made (ie 10^-2)] * [1000 uL / 1 mL] = CFU/mL

Results:

Raw Data:

Spore Stock D					8/17/14
	10^-2	0.002				AVG	StDv	CFU/mL	CFU /mL StDv
Colonies	19	26	22	12	16	19	5.39	850000	269258.2404
CFU/mL	950000	1300000	1100000	600000	800000
BHI
	No Heat
	10^-2	0.002				AVG	StDv	CFU/mL	CFU /mL StDv
Colonies	7	20	18	12	15	14.4	5.13	720000	256417.6281
CFU/mL	350000	1000000	900000	600000	750000
	Heat
	10^-3	0.0625				AVG	StDv	CFU/mL	CFU /mL StDv
Colonies	2	5	9	1	7	4.8	3.35	7680	5354.62417
CFU/mL	3200	8000	14400	1600	11200
H2O
	No Heat
	10^-2	0.002				AVG	StDv	CFU/mL	CFU /mL StDv
Colonies	16	22	21	13	14	17.2	4.09	860000	204328.1674
CFU/mL	800000	1100000	1050000	650000	700000
	Heat
	10^-2	0.002				AVG	StDv	CFU/mL	CFU /mL StDv
Colonies	14	16	19	12	11	14.4	3.21	720000	160468.0654
CFU/mL	700000	800000	950000	600000	550000
BAD
	No Heat
	10^-2	0.002				AVG	StDv	CFU/mL	CFU /mL StDv
Colonies	30	26	34	28	32	30	3.16	1500000	158113.883
CFU/mL	1500000	1300000	1700000	1400000	1600000
	Heat
	10^-2	0.002				AVG	StDv	CFU/mL	CFU /mL StDv
Colonies	30	22	26	33	36	29.4	5.55	1470000	277488.7385
CFU/mL	1500000	1100000	1300000	1650000	1800000
Spore Stock E
	10^-2	0.002				AVG	StDv	CFU/mL	CFU /mL StDv
Colonies	20	25	15	18	22	20	3.81	1000000	190394.3276
CFU/mL	1000000	1250000	750000	900000	1100000
BHI
	No Heat
	10^-1	0.02				AVG	StDv	CFU/mL	CFU /mL StDv
Colonies	20	24	15	27	18	20.8	4.76	104000	23822.2585
CFU/mL	100000	120000	75000	135000	90000
	Heat
	10^-0	0.2				AVG	StDv	CFU/mL	CFU /mL StDv
Colonies	2	1	0	1	3	1.4	1.14	700	570.0877125
CFU/mL	1000	500	0	500	1500
H2O
	No Heat
	10^-1	0.02				AVG	StDv	CFU/mL	CFU /mL StDv
Colonies	22	21	27	17	20	21.4	3.65	107000	18234.58253
CFU/mL	110000	105000	135000	85000	100000
	Heat
	10^-1	0.02				AVG	StDv	CFU/mL	CFU /mL StDv
Colonies	15	18	22	15	19	17.8	2.95	89000	14747.8812
CFU/mL	75000	90000	110000	75000	95000
BAD
	No Heat
	10^-3	0.0625				AVG	StDv	CFU/mL	CFU /mL StDv
Colonies	5	7	8	2	1	4.6	3.05	7360	4879.344218
CFU/mL	8000	11200	12800	3200	1600
	Heat
	10^-3	0.0625				AVG	StDv	CFU/mL	CFU /mL StDv
Colonies	2	5	6	5	3	4.2	1.64	6720	2629.068276
CFU/mL	3200	8000	9600	8000	4800
Spore Stock A*					*Old Data
BHI
	No Heat
		0.002				AVG	StDv	CFU/mL	CFU /mL StDv
Colonies	30	40	45	25	20	32	10.37	1600000	518411.0338
CFU/mL	1500000	2000000	2250000	1250000	1000000
	Heat
		0.002				AVG	StDv	CFU/mL	CFU /mL StDv
Colonies	1	1	2	3	0	1.4	1.14	70000	57008.77125
CFU/mL	50000	50000	100000	150000	0
H2O
	No Heat
		0.002				AVG	StDv	CFU/mL	CFU /mL StDv
Colonies	11	19	22	25	16	18.6	5.41	930000	270647.3721
CFU/mL	550000	950000	1100000	1250000	800000
	Heat
		0.002				AVG	StDv	CFU/mL	CFU /mL StDv
Colonies	14	22	12	20	21	17.8	4.49	890000	224722.0505
CFU/mL	700000	1100000	600000	1000000	1050000
BAD
	No Heat
		0.0002				AVG	StDv	CFU/mL	CFU /mL StDv
Colonies	4	7	5	2	3	4.2	1.92	2100000	961769.2031
CFU/mL	2000000	3500000	2500000	1000000	1500000
	Heat
		0.0002				AVG	StDv	CFU/mL	CFU /mL StDv
Colonies	1	4	7	3	5	4	2.24	2000000	1118033.989
CFU/mL	500000	2000000	3500000	1500000	2500000

I'm not exactly sure how to calculate the standard deviations for percent germination at this point, but will look into it. It appears there is minimal germination of P. larvae in H2O and BAD, but nearly 100% germination in BHI over the course of 2 hours incubation at 35C.

//EWW

The Waxworm & the P. larvae

From 8-14-14.

No other wax moths appear to have hatched from the eggs in the three treatment amounts of BAD on the 24 well plate. Perhaps they still need more time to incubate or they have already been killed (either by drying out, damage to the egg during transfer, drowning while trying to hatch, or other similar complications). I will continue to monitor their progress.

A diet was prepared for the wax worms once they hatch from their eggs. "Diet 3" was prepared according to COLOSS specification (Link). This was the simplest of diets I have found, plus all the ingredients present in the lab for its constitution. The recipe for Diet 3 is listed below:

DIET 3

7 parts dog food*

1 part sterile ddH2O

2 parts honey (Mel-O brand)**

*needs to be ground up prior

**Honey from Link, needed to be pre-warmed to dissolve any honey crystals that had formed in the bottle

Dog food was ground up using a mortar and pestle. The process was performed in small increments as the mortar was not very large. The smaller the amount the more thoroughly the homogenization could be performed. 

80 mL of honey was combined with 40 mL of ddH2O in 240 mL of ground up dog food in a small mixing bowl. The mixture was processed using a gloved hand until thoroughly homogenized. Approximately 150 mL of Diet 3 was transferred to a 500 mL container. 

All the waxmoths at 35C have died (most died within 1 week). The eggs that were adhered to the wax paper hanging from the covers were carefully removed either by VERY gentle abrasion if it looked like they were about to fall off or by cutting the paper around it. Then the small amount of wax paper containing the eggs along with any free eggs were transferred to the containers with 150 mL of Diet 3 (picture below). 

Four small air holes were punched into the cover each container. Containers were slightly mixed to cover the wax paper, this created a rugged terrain for the wax worms. Containers were incubated at 35C in the Fisher Lab. They will continue to be monitored for the emergence of the wax worms.

The left over Diet 3 was stored at 4C in the Fisher lab refrigerator.

The was moths presence in 30 C have not yet died and are still very active. It doesn't appear they have laid any eggs yet either. I will continue to monitor them as well.

//EWW

Probiotic & Antioxidant LC50

From 8-14-14.

Four additional honey bee larva are now dead. This brings the total dead to 7 out of 12 (58.3%) here on day 8. Usually on day 8 the larva have ingested all their food (the BAD) and have begun pre-pupation (straightening out), however none of the larva in this experiment have done so. Most that are still showing signs of life (slight undulation and reflective glistening skin) have not yet eaten all the present BAD. This is highly unusual, typically the larva eat every last drop that is present and are very active, moving their body and head around. These larva barely move. All of this leads me to believe that they are not well and could either be succumbing to P. larvae or the yeast that was spiked into their BAD on day 0.

Additionally, on some of the confirmed dead larva today the presence of small white dots could be seen on their surface (or just below their surface as it is translucent).

I plan to homogenize each dead larva individually and determine the presence of  P. larvae through PCR using the AFB - F/R primer sets in the near future.

//EWW

Probiotic & Antioxidant LC50

From 8-12-14.

On August 13th (day 6) - Each larva was fed an additional 50 uL of BAD. One of the larva appears to be dead. This brings the total dead to three out of 12 (25%) at day 6.

On August 14th (day 7) - Honey bee larva do not appear to be in good health. None are as active as they should be on day 7 as per previous experiments. A large amount of BAD still remains in most of the wells. Additionally, it is difficult to distinguish if they are dead or alive as they only slightly ungulate. Normally on day 7 the larva are their most active- having eaten all their food and beginning pre-pupation stage.

I will continue to monitor their progress.

USDA Hives
Unfortunately two of our Jenter Boxes at the USDA have been damaged beyond use. They were submerged in boiling water for a long period of time in order to facilitate detachment of adhered wax and other debris.  The high temperature was apparently too much for the plastic boxes and they meleted and warped. This will further hamper our collection of honey bee larva.

Warped Jenter Boxes

The amount of larva we are able to recover seems to have decreased by 80-90% from what we were able to recover from the same hives in July. It appears the queen bee is laying eggs in nearly every one of the wells, however they are not being taken care of by the worker bees and are either left to dry out, taken away, or eaten. By the fourth day (when we'd collect the box) nearly all of the eggs/hatched larva are missing.

//EWW

The Waxworm & the P. larvae

From 8-12-14.

The single hatched waxworm appears to be dead. It was surrounded by BAD and now looks like it is either dead or in some sort of cocoon. None of the other eggs in the BAD has hatched yet and maybe wont. Some of the eggs appear to be broken open and a small unknown substance is located right next to it. This likely indicates that the eggs hatched, but the waxworm died shortly after possibly drowning in the BAD that encircled it.

//EWW

Probiotic & Antioxidant LC50

From 8-8-14.

On August 9th (day 2) - transferred each larva to a new well in the 24 well plate and fed each larva 10 uL BAD. There doesn't appear to be any deaths at this time point, although it is more difficult to determine at this early instar due to a general lack of movement and glistening caused by the BAD.

On August 10th (day 3) - Fed each larva an additional 20 uL of BAD. Identified one dead larva out of the 12.

On August 11th (day 4) - Fed each larva an additional 30 uL of BAD. No new deaths were observed in any of the larva.

On August 12th (day 5) - Each larva was fed an additional 40 uL of BAD. An additional death was observed. This takes the total dead to two larva out of 12 (16.7%).

These results so far are interesting. According to my finding in the P. larvae LC50 assay on 8-3-14 the first instar honey bee larva that were fed 1000 CFU/mL of P. larvae spores displayed about 50% death by day 5. Here in this experiment there is only 16.7% death after 5 days. In this experiment the first instar larva were fed 1000 CFU/mL of P. larvae spores (from the same stock as the LC50 assay) along with 1000 cells/mL of yeast. Could it be that the yeast had some sort of inhibitory effect on the spores or their virulence? I will continue to monitor survival.

This experiment with the spores/yeast was my attempt at salvaging my initial attempts after discovering a significantly less amount of available honey bee larva. This is an excellent preliminary glimpse, however this experiment should obviously be repeated with a larger sample size and controls!

//EWW

The Waxworm & the P. larvae

From 8-4-14.

I observed that a single waxworm has hatched from its egg in the 5 uL BAD treatment group. It is only about 2 -3 mm in length, but is visible with the naked eye. It doesn't appear that it has removed any of the present 5 uL BAD, but is very active on the side of the well moving around. I put an additional 10 uL of fresh BAD next to it and will continue to monitor its growth.

None of the other eggs have hatched yet. I will also need to create food for the other waxmoth eggs in order to continue to maintain a healthy colony.

//EWW

Probiotic & Antioxidant LC50

From 8-7-14.

Was all prepared to begin experiment, however there was significantly less larva recovered from the Jenter box at the USDA hives than initially anticipated. Just three days ago nearly the entire box was full of eggs, however today there was barely a dozen larva visible in the box. How odd. I am 100% positive that the Jenter box was full of eggs when I freed the queen three days ago. What few 1st instar was present, I transferred to a 24 well plate and fed 5 uL of Treatment F.

Larva were incubated at 35C in anaerobic chamber.

Special Note: I have been stung twice today by honey bees. Once through my glove on my right middle finger. And the other was INSIDE my right nostril. I was alone tending to the bee hives today. As I was driving back to the USDA there were a number of bees still in my vehicle. I removed my bee keeping veil for fear of being pulled over. Typically having the windows down would deter any bees from taking flight and attacking, however as I slowed down while passing road construction they took flight and attacked. The pain was unimaginable. Took antihistamine and applied hydrocortisone cream about two hours later.  Symptoms include severe head ache, nostril pain on right side, jaw pain on right side, and an overall feeling of unwell. 

//EWW

Probiotic & Antioxidant LC50

Goal: Perform the LC50 P. larvae assay using honey bees using different treatment groups. One will be a probiotic group and another will contain an antioxidant. Details to follow.

Red Star Active Dry Yeast (Link) was obtained. Several particles of yeast were aseptically spread onto LB Only agar and incubated at 30C. Isolated colonies were transferred to new LB Only agar and continued to incubate. A total of 20 mL of 10 mM Tris was added to the surface of an LB Only plate containing yeast growth (plate seen below). Tris was added in 10 mL increments, plates were scraped using L-shaped cell spreaders, and solutions were pipetted off the surface and transferred to a 50 mL conical tube.

Yeast on LB

A small portion of the  Yeast Stock was diluted 1:100 in ddH2O and 10 uL were transferred to a hemocytometer for quantification. Cells were counted and averages were determined (48 cells per small square).

Hemocytometer Equation:

Cells Counted * (dilution factor / number of small squares counted) * 10,000 = Cells / mL

or

Average cells per small square * dilution factor * 10,000 = Cells / mL

Yeast stock was determined to be at 4.8x10^7 cells/mL.

There will be six different treatment groups for the upcoming experiment:

A. BAD only
B. BAD + 1000 cells/mL Yeast
C. BAD + Antioxidant
D. BAD + 1000 CFU/mL P. larvae
E. BAD + 1000 CFU/mL P. larvae + Antioxidant
F. BAD + 1000 CFU/mL P. larvae + 1000 cells/mL Yeast

BAD + Antioxidants (Treatment C) was provided to me from the USDA as diluted by Garret S. for Anna B.'s antioxidant/wax project.

Dilutions:

Treatment B: Yeast Stock was diluted 1:10 down to 4.8x10^4 cells/mL in ddH2O (100 uL + 900 uL   H2O). 11 uL of this 10^4 dilution was added to 489 uL of BAD to create 1056 CFU/mL Yeast spiked BAD treatment group

Treatment D: P. larvae Spore Stock A (3.05x10^6 CFU/mL) was diluted 1:10 down to 3.05x10^4     CFU/mL in ddH2O (100 uL + 900 uL H2O). 16 uL of this 10^4 dilution was added to 484 uL of BAD to create 976 CFU/mL P. larvae spiked BAD.

Treatment E: Same procedure as Treatment D, except BAD containing the antioxidant was used.

Treatment F: 11 uL of the 10^4 Yeast dilution (Treatment B) and 16 uL of the 10^4 P. larvae dilution (Treatment D) were added to 473 uL of BAD.

Dilutions were stored at 4C overnight. They will be applied to 1st instar honey bee larvae in about 16 hours after creation.

//EWW

P. larvae CFU/larva Pilot

From 7-31-14.

PCR performed on several colonies recovered from homogenized honey bee larva. AFB-F/R primers used. Loaded amplicons (2uL EZVision + 7uL DNA) into a 1% gel. Ran electrophoresis at 120V for 0.5 hour.

There was no amplification of any of the colonies recovered from the homogenized larva tested positive using the AFB primers (Gel not shown). Positive control checked out. It could be that whatever the bacteria colony was outcompeted the slow growing P. larvae. I am not sure how to proceed with this study at this moment. It will likely be put on hold for the time being to focus on more... lucrative experiments.

//EWW

The Waxworm & the P. larvae

From 8-3-14.

Methods paper for rearing waxworms/moths in vitro from COLOSS.

Small clusters of waxworm eggs were discovered on the folded waxpaper from a container created on 8-3-14. Eggs were carefully teased apart and transferred to a 48 well plate using a camel hair paint brush and sharp forceps.

12 eggs were transferred to 2 uL of BAD
6 eggs were transferred to 5 uL of BAD
6 eggs were transferred to 10 uL of BAD

48 well plate was incubated at 35C in anaerobic chamber with ~90% humidity. I will monitor their progress daily.

//EWW

P. larvae LC50

From 7-25-14.

Completed LC50 study (day 9). 

Raw Data

DEAD
	Day 1	Day 2	Day 3	Day 4	Day 5	Day 6	Day 7	Day 8	Day 9
Control	5	1	2	1	1	0	0	0	0
400	9	2	2	3	1	1	0	1	0
1000	10	7	4	2	3	5	0	1	2

Mortality
	1	2	3	4	5	6	7	8	9
Control (45)	0	2.5	7.5	10	12.5	12.5	12.5	12.5	12.5
400 CFU/mL (44)	0	5.26	10.53	18.42	21.05	23.68	23.68	26.32	26.32
1000 CFU/mL (42)	0	21.875	34.375	40.625	50	65.625	65.625	68.75	75

//EWW

The Waxworm & the P. larvae

Continuation of 8-1-14.

Several of the more of the waxworms that have been incubating at 35C have emerged as waxmoths. None of the waxworms at 30C have emerged and most appear to be dead (black in color). Emerged waxmoths are pictured below.

                                            

Emerged waxmoths

Five waxmoths were transferred to three 500 mL containers (total of 15 waxmoths) with folded wax papers hanging from the caps (pictures below).  Containers are incubating at 35C.

//EWW

The Waxworm & the P. larvae

From WaxWorm Notes on 7-22-14.

Waxworms have been incubating at two different temperatures for 11 days. Some of the waxworms that have been incubating at 35C have emerged as waxmoths. None of the waxworms that had been incubated at 30C has emerged as waxmoths. In fact, several of the waxworms that had been incubated at 30C have turned black and died. Most of the waxworms incubated at 35C are pupating (look burnt brown indicating a cocoon).

Three waxmoths were seen in the container of waxworms incubated at 35C. Picture seen below.

Additionally, a single waxmoth was retrieved from the USDA on one of their honey comb frames (from Anna B's experiment, termed the "old" wax). This waxmoth was transferred from the USDA building to NDSU in a 50 mL conical tube and transferred to a 500 mL container like the one above. 

Dixie brand Kabnet Wax paper (Link) was cut into long thing strips and folded up accordion style and placed inside the 500 mL container that housed the waxmoth. Three waxmoths from our own colony that had recently emerged were also transferred to this container. The waxmoths were transferred using a long forceps with wax paper taped onto it (pictures below).

The hopes of combining the wild type waxmoth from the USDA hives and our own colony waxmoths is that they will breed and increase genetic diversity in our own colony.

Two folded strips of waxpaper were hung from the cap of the 500 mL container housing the waxmoths (pictures below). The hope is that the waxmoths will lay eggs in the folds of the waxpaper and I can easily retrieved them each day. I will monitor how well this process works after 24 hours of incubation at 35C.

//EWW