Red Flour Beetle Maintenance

From 9-11-14.

Checked on my colony of red flour beetles (RFB). It has been 270 days (8 months, 28 days) since the last time the RFB had their food source changed. I have not checked on them since then. I varied the type of flour they were provided and observed some interesting results.

There were three different treatment groups in this "experiment". RFB were provided either corn flour, wheat flour, or a 50:50 mix of both. Treatments were in quadruplicate in glass masons jars with a cheese cloth cover. The jars were incubated at room temperature in a box protected from light for the 270 days. There were about 200 - 300 RFB per jar.

Live RFB that are being transferred

After 270 days, all of the RFB in all four of the corn flour treatment groups were completely dead. All of the wheat flour treatment group RFB were alive and well. And two of the four jars in the 50:50 mix treatment group were completely dead and the other two were alive and well.

Live RFB that are being transferred

Results:

Treatment	replicates	After 270 days
Corn Flour	4	All 4 dead
Wheat Flour	4	All 4 alive
50:50 Mix	4	half (2) jars dead

Dead RFB in the corn flour treatment group

Discussion:

It would appear that the RFB do not do well with only being provided the corn flour as a long term food source. All of the 200 - 300 RFB in the four replicate groups in the corn flour treatment group were completely dead; not moving, some upside down. Perhaps it was the nutrient difference in the corn flower that lead to the death of the RFB in this treatment group. Or perhaps the RFB utilized the nutrient source more quickly than in the other treatment groups. It appeared that the number of dead RFB was relatively equivalent to what was initially put into the jars.

The RFB seemed to do very well on the wheat flour after the extended period of time. All of the replicates had lively and active RFB in them. What is most interesting is that half of the replicates in the 50:50 mix group were completely dead. It is interesting since half the contents of the 50:50 mix (the corn flour) was related to the death of their entire treatment group!

The RFB from the wheat flour treatment group were transferred to fresh wheat flour in the mason jars. Of the two jars that had surviving RFB in the 50:50 mix treatment group, half were transferred to two jars containing the 50:50 mix again, and the other half were transferred to two jars containing corn flour only. It would be interesting to see if by exposing the RFB to the 50:50 corn:wheat flour mix that they can now survive on the corn flour only diet. I'll check the survival of the RFB from time-to-time (hopefully sooner than 270 days from now).

//EWW

Sporulation on Blood Agar

From 5-4-15.

Two isolation streaks of P. larvae 9545 (from the -80C stock) were struck out onto MYPGP agar plates and incubated at 37C for three days until nice isolated colonies formed. A single isolated colony was inoculated into 10 mL of BHI broth and incubated at 37C 225 rpm for 24 hours.

I made more Columbian blood agar plates (Columbian agar base + 5% defibrinated sheep's blood). I am currently out of fresh defibrinated sheep's blood. I made plates and not slants this time since it appears that I can recover a similar concentration of spores, if not a higher titer, from the plates. Plus, I was having issues with using the slants with accidentally removing agar from them and transferring them to the Eppendorf tubes during the spore extraction procedure.

A 100 uL volume of the overnight culture was inoculated and spread onto the blood agar plates. The plates were incubated at 37C 5%CO2 in the media kitchen (Van Es Room 115) incubator was used. The plates will incubate for seven days until the spores will be extracted and purified.

//EWW

Wax Worm/P. larvae LD50

From 5-25-15.

I would like to see if the PH agar can be used to propagate newly hatched wax worms. A small clutch of wax worm eggs were placed in the center of PH agar plates, wrapped in Parafilm, and incubated (not inverted) at 30C.

Large petri plate with PH agar with small clutch of WW eggs in the center

Unfortunately, there was condensation on the PH agar plates and the eggs were underwater after 24 hours. The plates were dried in the biological safety cabinet, but I'm not sure if the eggs will survive. The plates were continued to incubate at 30C, however a number of the plates showed signs of fungal contamination after 48 hours. This is likely a result of the microwaving that was done to sterilize the media. I'm continuing to monitor the eggs on the agar to see if anything hatches.

Additional PH agar were made in order to repeat this study:

PH agar (125 mL)

2.5 g Agar

1.25 g Potato Flakes

2.5 g Honey

Agar was microwaved for a longer and more thorough period of time. PH agar was poured into small petri plates. The plates were left in the biological safety cabinet to remove condensation.

PH agar in small petri plates

Plates were left at room temperature to monitor for fungal growth before use.

//EWW

ClO2- Relative Humidity in Chambers

Goal: After discussion with David S. and Joel T. the direction of altering the relative humidity inside the modified anaerobic chambers was brought up. Joel was very happy with the killing curves I was getting, but remarked that one of the biggest factors facing the actual application and use of the chlorine dioxide gas in the field is the humidity in the area. Some places are rather dry and others are very humid, so for my next series of experiments I will alter the humidity inside the chambers and then generate ClO2 inside them. The effect the gas has on the P. larvae spores will be interesting to see. Since the 200 mg total dry weight of ClO2 reagent seemed to have a large effect on the spore survival, that is the weight that will be used throughout these experiments.

I will start by getting an idea of the ppm of chlorine inside the chambers over the course of six hours at a very low relative humidity. For this, no additional water was added to the chambers which resulted in a relative humidity inside the chambers of around 30-35%.

Reading the Cl2 ppm columns

An image of what the columns look like after taking a 200 mL sample from the modified anaerobic chambers with ClO2 reagents in them. There is a green "front" in the columns as you take a gas sample.

The ppm were determined by reading the spot right at the interface between the green front and where the stronger orange color begins. This spot is illustrated by an arrow in the image above. Calipers will be used to more accurately determine the ppm.

Relative Humidity Estimates

A rough estimate of the amount of water in the air inside the chambers was determined based off of this graph.

relative humidity	g of H2O /kg air	g H2O in air/ chamber
30%	5	12.5
60%	9	22.5
90%	11	27.5

//EWW

Wax Worm/P. larvae LD50

From 5-14-15.

I've experimented with a new type of Potato-Honey agar to potentially be used as a media for the P. larvae LD50 assay. The recipe was found on an online blog from some guy who raises wax worms in his basement.

Potato-Honey (PH) Agar (1 Liter)

20 g Agar

10 g Instant Potato Flake

20 mL Honey

(Sterilize in Microwave)

A volume of 125 mL of PH agar was made in a 250 mL flask. The solution was microwaved until the potato flakes dissolved and became homogenous (or close to it). The solution was periodically removed from the microwave and stirred by hand. After the PH agar was microwaved a small portion was transferred to the wells of a 96 well plate using a sterile eye dropper. This method seemed to work well and the agar solidified nicely in the wells. My only concern is how sterile the PH agar is and if something will begin to grow in the agar. Remaining PH agar was poured into petri plates, it'd be interesting to see if WW eggs would hatch on this surface...

Side view of 96 well plate with PH agar in the wells

Bottom of 96 well plate with PH agar in it

PH agar is very dense and heavy compared to other agar types (like Nutrient Agar or LB Agar). It isn't as soft and has a rather solid consistency thanks to the presence of the honey. 

//EWW

Wax Worm Maintenance

From 5-5-15.

Wax moth eggs were transferred to new containers with diet. Eggs were present on the tops of the containers near where the lid of the container attaches. A number of the eggs were present on the wax paper at that interface. A hodgepodge of containers were used for new eggs. Most of the wax worms have almost completed pupation and are laying eggs, so I'll continue to monitor and transfer eggs as I see them.

Contamination:
There were two large secondary containers each with four smaller containers within. The containers were stored in the 30C walk in incubator, but the container that was stored in the secondary corner of the room had three of its four smaller containers contaminated with fungus! The other secondary container that was in the middle of the shelf had no fungal growth. Not a big problem with the survival of the colony as there are still PLENTY of eggs in the other containers. In fact, some of the eggs were stored in an Eppendorf tube at 4C.

It would appear that conditions were different enough in the 30C incubator depending on where the containers were that it caused fungal growth. In the future, I will only store the containers in the middle of the shelf just to be safe.

//EWW

ClO2 generation in chambers

From 5-17-15.

Checked the ppm of Cl that was generated inside the modified anaerobic chambers using a total weight of 100 mg of ClO2 reagent. Hours 3.5 to 6.0 were checked and will be added to what was previously discovered.

Time	ppm Cl
0.5 hr	65
1.0 hr	75
1.5 hr	90
2.0 hr	100
2.5 hr	105
3.0 hr	105
3.5 hr	100
4.0 hr	100
4.5 hr	95
5.0 hr	95
5.5 hr	90
6.0 hr	80

As was predicted, based on the previous results, chlorine present in the air inside the chambers peaks and then drops off over the six hours. The peak appears to be around 2.5 - 3.0 hours. The peak ppm of Cl was about 105 and the presence of ClO2 gas inside the chamber is about 0.25 of that (0.25 is about the conversion factor that was determined by David S.). The presence of ClO2 inside the chambers is incredibly low, which it is even more surprising that the spore killing rate is so high. 

Below are the calculations received from the USDA regarding ClO2 gas generation inside the modified anaerobic chambers: 

Assumptions:

Chlorine Dioxide	67.45 g/mol
Temperature	25 C (298.15 K)
Pressure	1 Atm
Total Volume	2.5 Liter
Release Media	10 mg ClO2 per 6 hours at 1 g each of Part A and Part B

Target Concentration	Total Mass ClO2		Concentration ClO2	Mass of Media Part A             Part B
μg/L	μg	mg	ppmv	mg	mg
10	25	0.025	3.6	2.5	2.5
50	125	0.125	18.1	12.5	12.5
100	250	0.250	36.3	25.0	25.0
200	500	0.500	72.5	50.0	50.0
250	625	0.625	90.7	62.5	62.5
500	1250	1.25	181.4	125	125
1000	2500	2.5	362.7	250	250
2000	5000	5.0	725.4	500	500
4000	10000	10.0	1450.9	1000	1000
5000	12500	12.5	1813.6	1250	1250

According to our initial predictions, with a total dry weight of 100 mg ClO2 reagents, we expected to see a ClO2 ppmv of 72.5 (note: not ppm of Cl). The amount of ClO2 we're actually generating inside the chambers is way lower than that (<20 ppmv). The reduced concentration is probably attributed to the polystyrene containers that are used along with the presence of the glass slides lining the bottom. These types of materials all serve as sinks for the gas and the above calculations depict the unrealistic ideal conditions.

//EWW

ClO2 Study - P. larvae Spores on Glass

From 5-15-15.

I was able to count CFU from the MYPGP plates that had the spores that were exposed to ClO2 gas for three hours on them. Spore stock #10 (1.1x10^7 CFU/mL) was initially inoculated onto the glass cover slips. Since a volume of 100 uL was added to the cover slips that means there is a concentration of 1.1x10^6 CFU on them before they were exposed to ClO2.

ClO2 Reagent	CFU/mL
0 mg	9.33E+05
50 mg	6.67E+05
100 mg	5.67E+05
200 mg	3.67E+05

There is a decreasing trend seen in spore survival as you increase the concentration of ClO2 reagents. This is a similar trend to what has been seen before after exposing for 6 hours, however the log killing was higher (due to the increase amount of exposure time). The standard deviations aren't very good, likely due to subtle decreases in spores between concentrations. For example, when 200 mg were used to treat spores on glass for 6 hours there was over a 3 log killing rate. It would appear that the length of exposure time has an effect on killing of spores, as well as generation of gas.

//EWW

ClO2 generation in chambers

Goal: Determine the rate at which ClO2 gas is being generated inside the modified anaerobic chambers. This information will be useful information for my ClO2 exposure study as an effective was of killing P. larva spores on apiary-related surfaces. Based on preliminary results, there appears to be a parabolic trend associated with gas generation inside the chambers, with Cl ppm peaking around 3 hours.

For this study, the modified anaerobic chambers were set up like they would be for the spore-exposure study (50 mL H2O, stir bar, etc.). A total weight of ClO2 reagents of 100 mg (50 mg from each Part A and Part B) was used for these experiments. The reagents were mixed inside a PCR tube and after mixing the tube was placed, open, inside the chambers. The ppm of Cl was checked every 30 minutes and were recorded.

Time	ppm Cl
0.5 hr	65
1.0 hr	75
1.5 hr	90
2.0 hr	100
2.5 hr	105
3.0 hr	105

I will continue the experiment with time points after 3.0 hrs up to 6.0 hours. 

//EWW

ClO2 Study - P. larvae Spores on Metal

From metal on 5-13-15.

I am able to count the CFU on the MYPGP from the spores on the metal coupons that were exposed to ClO2 gas for three hours. Unfortunately, I wasn't able to count any colonies from the treatment groups (even from the 10^-0 dilution!) as no colonies grew on the plates.

Spore stock #11 was added to the coupons, which means there was about 1.8x10^5 CFU on the coupon when it was exposed to the ClO2 gas. I was able to count CFU from the spores on metal that were exposed to ClO2 gas, unfortunately it was 2.2x10^4 CFU/mL, which means there was only about a 12% rate of recovery of the spore from the coupons! This was a similar problem that I had with the spores on wood, the spore recovery is very poor. I will need to adjust my re-suspension protocol for these two surfaces (pine chips and metal coupons). Perhaps increasing the time the surfaces are soaked in the 1 mL H2O would help with re-suspension.

Below is an image of one of the MYPGP dilution plates. There were only two colonies on the plate from the 10^-0 dilution. However, duplicate plates did not have any colony growth on them.

Spores on metal exposed to 50 mg weight of ClO2 reagent for 3 hours 

I will repeat this experiment in the future and adjust the re-suspension procedure.

//EWW

ClO2 Study - P. larvae Spores on Glass

From 5-10-15.

Spore stock #10 (1.1x10^6 CFU/mL) was used in a ClO2 exposure experiment for three hours on glass cover slips. A volume of 100 uL was added to sterile glass cover slips as before and they were exposed to ClO2 also as previously done, however, the exposure time was not six hours as before, but only three hours this time.

Three different ClO2 concentrations were used in this three hour exposure experiment: 50 mg, 100 mg, and 200 mg. A control of no ClO2 exposure was also used.

The ppm of chlorine gas in each container was checked using the columns as before by taking a 200 mL volume of air sample out after three hours. Interestingly, the ppm of Cl gas seem to agree with what was observed previously the last time this experiment was ran for only three hours. The ppm were about twice what was seen after six hours. This further indicates that there is likely a parabolic trend with generation of ClO2 gas inside the modified anaerobic chambers.


//EWW

ClO2 Study - P. larvae Spores on Wood

From wood on 5-10-15.
From metal on 5-13-15.

I was able to count colonies of P. larvae that grew on MYPGP agar. These spores were exposed to ClO2 gas on pine chips for six hours.

How to calculate CFU/mL
(CFU * Dilution Factor * Volume Factor)
or
(# CFU) * ( 1 / 10^ dilution factor) * ( 1 / volume plated in mL) = CFU / mL
ex:
3 * ( 1 / 10^ (-4)) *  ( 1 / 0.01mL) = 3x10^6 CFU /mL

ClO2 conc	CFU/mL	CFU/mL
0 mg	14000	1.40E+04
25 mg	9000	9.00E+03
50 mg	950	9.50E+02
100 mg	200	2.00E+02

Spore stock #14 was added to each pine chip. 1.6x10^6 CFU/mL was the concentration of this spore stock, which means there was 1.6x10^5 CFU added to each pine chip. That also means there was only about a 8.75% spore recovery from the pine chip that wasn't exposed to any ClO2 gas, which is terrible. Ideally, >90% recovery would be preferable. 

I attribute the poor spore recovery in part to the pine chips becoming saturated and portions of the spore stocks soaking through and onto the petri plate where it was drying. Another possible contributor could be due to the properties of the pine chip with retaining the spores once re-suspended. I will have to alter my procedure to account for these possibilities. Either by added smaller volumes of spores at a time and allow them to dry before adding the entire volume. I may also try to locate some thicker pine chips to use that wont become saturated as quickly with only 100 uL volume.

Also, there appeared to be a lot of different colony morphologies present on the MYPGP agar plates, more than usual for P. larvae. It could be related to other endospores already present in the pine chips that survived the autoclave sterilization process.

//EWW

Wax Worm/P. larvae LD50

From 5-8-15.

I added wax to three 96 well plates. Bee's wax was cut to small shavings using a sterile scalpel in a petri dish. Every pre-caution was taken to not contaminate the wax beyond its natural state. The wax itself is not sterile, but I didn't wish to add to it! The wax shavings were transferred to the 96 well plates using a sterile forceps.

Only the first and last four columns of each plate had wax added to them. This is because when the wax worms will be added (and an adhesive cover added) it will be easier to access them if they are not in the middle of the plate. The plates were heated in a ~65C water bath for ~5 minutes or however long it took to melt the wax to the bottom of the plate.

Hot plate water bath used to melt the wax in the 96 well plates. Notice, only the first and last 4 columns have was in them.

Bottom of 96 well plate after wax had melted. Only enough wax was added to cover the bottom of the well. In this case, less wax was preferable to more.

The plates were stored at room temperature. The spores have yet to be added to the wax, along with the early instar wax worms.

//EWW

ClO2 Study - P. larvae Spores on Metal

Spores on glass from 5-10-15.

Beekeepers use a number of metal tools when maintaining their hives (crowbars, scrapers, shovels, spades, smoke canisters, etc). The efficacy of ClO2 gas as a disinfectant of P. larvae spores on metal will be determined. I acquired 500 cut stainless steel coupons from NDSU shop foreman Kyle R. The coupons are made of 301 grade steel cut to 0.5 inch squares. P. larvae 9545 spores will be used in this experiment initially, followed by the North Dakota isolates.

100 of the metal coupons were autoclaved using the dry cycle in order to sterilize them. Spore stock 11 (1.8x10^6 CFU/mL)was used for this experiment. A 100 uL volume of spore stock was added to the tops of the sterile metal coupons. The spores were allowed to adhere to the surface of coupons by incubating at room temperature for 1 hour in a biological safety cabinet. 

100 uL of spores on metal coupons

The experiment was set up as before, with three coupons being placed inside a single modified anaerobic chamber. Total dry weights of chlorine dioxide reagents were mixed in PCR tubes and the reaction was allowed to take place for three hours this time. This is a change from the previous experiments that ran six hours.

After three hours, the concentration of chlorine gas was determined using the columns once again. 

Chlorine concentration in each container after 3 hours. The numbers indicate what the total weight of reagents were (in mg)

Dry wt	Conc Cl
0 mg	0
50 mg	50 ppm
100 mg	100 ppm

The detected ppm of chlorine was very surprising when compared to what has previously been seen using the glass cover slips at 6 hours. The 50 mg concentration of chlorine was pretty close to what had been previously seen, but the 100 mg concentration resulted in twice the detected ppm of chlorine gas after only three hours compared to the previously performed six hours. It could be that the gas peaks earlier than six hours and then lows due to exposure to the 50 mL of H2O being present. I could conduct an experiment of the gas generation inside the chamber in the future, but for now I will just continue with these experiments and see if this new trend continues.

The metal coupons treated with ClO2 gas were re-suspended in 1 mL of sterile ddH2O. The coupons were allowed to incubate at room temperature in the water for 5 minutes before diluted in H2O and plated on MYPGP agar. The plates were incubated at 37C for four days before colonies can be counted.

//EWW