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