Sporulation on Blood Agar

From 4-25-15.

It has been ten days that the P. larvae 9545 has been incubating on the blood agar slants at 37C. Today I extracted the spores from the slants as previously performed (washing, heating, and centrifuging). Five slants were consolidated together into one spore stock.

One of the dilution plates from the spore stocks

I was also able to quantify the CFU/mL of the P. larvae spore stocks that were from the blood agar plates. Below is there determined concentration:

Stock #	Source	CFU/mL	CFU/mL
17	Plate	1600000	1.6x10^6
18	Plate	3600000	3.6x10^6

Other spore stocks here.

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

//EWW

ClO2 Study - P. larvae Spores on Glass

From 4-23-14.

I was able to count the P. larvae colonies that grew on the MYPGP agar plates after being exposed to 12.5 mg of ClO2 reagent.

The CFU/mL was calculated using the formula:

One of the MYPGP dilution plates with the P. larvae on it that had been exposed to 12.5 mg of ClO2

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

The data is stored in an Excel file.

Sporulation on Blood Agar

From 4-18-15.

It has been seven days that P. larvae ATCC 9545 has been incubating on blood agar at 37C with 5% CO2. The spores were extracted from the plates as previously performed (washing and heating at 60C). The newly created spore stock #17 and #18 were diluted and plated on MYPGP agar for colony counts. The plates were incubated at 37C and will remain there for three days until colonies form.

//EWW

Detection of P. larvae in Local Honey

From 4-22-15.

The plates that had been struck with the P. larvae isolates from local honey samples had been incubating for three days at 37C and now have good colony formation. Interestingly, there was no growth on the LB agar plates and each isolate had growth on the MYPGP agar. As the isolates are P. larvae, they should grow well on MYPGP agar.

Isolates stuck out on MYPGP & LB agar

dd1-5a

dd5-4a

dd8-55a

dd11-2a

dd12-22a

Crude genomic lysate was made of each isolate and will be used as template DNA for a PCR reaction using the AFB primers to confirm that they are (most likely) indeed P. larvae.

An isolated colony from each isolate was inoculated into 10 mL of BHI broth and incubated at 37C shaking 225 rpm. I plan to inoculate a broth culture of these isolates onto Columbian blood agar slants and extract their spores. These spores will be used in my chlorine dioxide study. I also plan on amplifying the 16S gene's of these isolates and send them in for sequencing.

//EWW

Germinant Receptors in P. larvae - Bioinformatics

From 4-18-15.

I've figured out how to correctly convert all my .sra sequences to .fastq files using a short script (macro). My previous attempt to convert the files was indeed incorrect. I created a short script in TextWrangler (just a text/notepad basically) and saved it as a .txt format. The name of this created script was called "Convertfastq" and contained this:

for f in *.sra

do

/Users/Elliott/sratoolkit.2.4.5-2-mac64/bin/fastq-dump --split-files $f -O /Users/Elliott/ncbi/public/fastq

done

The Convertfastq.txt file was saved in the same place where all my .sra files were located (ncbi/public/sra).

In the terminal, I navigated to the sra directory and type this command in to allow me access to use the created Convertfastq.txt script 

$ chmod 755 Convertfastq.txt

While in the directory containing the Convertfastq.txt file I typed in the command to execute the script. 

$ ./Convertfastq.txt

Which took about about 2 hours to complete. All the newly created fastq files were moved to the new directory in ncbi/public/fastq

//EWW

ClO2 Study - P. larvae Spores on Glass

From 4-19-15.

Repeated ClO2 exposure to P. larvae spores. A lower concentration of ClO2 reagents was used in this experiment. Spore stock #16 was used for the adhesion onto glass cover slips. The experiment was performed exactly as before (same setup) except a total weight of 12.5 mg of ClO2 reagent was used (~6.25 mg each). The reagents were mixed inside PCR tube as before and, surprisingly, appeared to mix well with each other.

I also performed another humidity & temperature check inside the modified anaerobic chambers after 6 hours.

Temperature, Humidity

28.7C, 96% (today)
28.8C, 95% (today)

28.8C, 96%(from 4-19-15)

The temperature and humidity inside the modified anaerobic chambers appear to be consistent between days, different chambers, and the different magnetic stir plates. 

Below is a graph of the cumulative results so far with the spores on the glass surface. I will know the CFU/mL of spores after they've been exposed to 12.5 mg of spores after this experiment today.

After the six hours have been reached using each different weight of ClO2 reagent I have taken a 200 mL sample using the the Cl columns to detect the ppm of Cl gas inside the chamber. Below is graph and data table containing the determined ppm of Cl for each dry weight of reagents used.

Weight	Cl ppm
200 mg	75
100 mg	55
50 mg	45
25 mg	25
12.5 mg	15
0 mg	0

//EWW

Detection of P. larvae in Local Honey

From 4-19-15.

Since I am apparently having so many issues with amplifying the 16S region of the selected P. larvae honey isolates I have decided to go back to the drawing board and re-streak out the cultures from freezer stock and try again. I did not use the same exact isolates as last time in all cases, but I did have representatives from the five different honeys that were screened and found to contain P. larvae. 

Isolates stuck out on MYPGP & LB agar

dd1-5a

dd5-4a

dd8-55a

dd11-2a

dd12-22a

The dd11-2a and dd12-22a are the same isolates previously examined as they were the only representative for their honey source that was seen to have an amplification using the AFB primers. 

//EWW

Detection of P. larvae in Local Honey

From 4-18-15.

Ran AFB PCR using the same DNA template previously used in the attempted 16S amplification attempt. The results were less than desirable.

The top lanes are the AFB amplicons and the bottom is the 16S amplicons. Lane labels are below the images. The two gels are the same, the one on the right it just overexposed so I could see it more clearly here.

Lane 1	Lane 2	Lane 3	Lane 4	Lane 5	Lane 6	Lane 7	Lane 8
Hi Lo	2a	22a	8a	63a	Q	RT	Pl

A volume of 5 uL of amplicon and 2 uL of EZ vision were loaded into each of the lanes. Not all the isolates amplified when using the AFB primers. All of them them except the roach trachea "RT" isolate should have! The positive control, P. larvae 9545 "Pl", checked out though. As you can also see, there was no bands that appeared using the 16S primers, however there was a lot of DNA in the wells. Perhaps I am overloading the lanes? 

Working on a solution to these issues...

//EWW

Wax Worm/P. larvae LD50

From 4-18-15.

Unfortunately, some of the wax worms were able to escape the 96 well with the transparent cover, but not nearly as many as before when using the metal cover. It is still somewhat difficult to determine if the wax worm  in each well is alive or dead as sometimes they are indeed alive, but just not moving at the moment (and there are 96 of them to count!). So, I've taken to marking the well with a colored marker if they are dead and I will use a different marker each day. An "X" will denote dead and an "O" will represent that the well was empty.

I set up two additional LD50 experiments in the same way that the last one was designed. Early instar wax worms were placed into the wells of a 96 well plate containing a piece of oat with endospores. The wells were sealed with the transparent film once again and a small hole was pierced in it for each well. The plates are incubating at 37C and I will monitor survival each day.

//EWW

ClO2 Study - P. larvae Spores on Glass

From 4-18-15.

I was able to accurately count the number of colonies that have formed on the MYGPG agar plates from the last ClO2 experiment with the P. larvae spores on glass. All the raw data is stored in an Excel file.

Average values:

CFU/mL
ClO2 conc	Dilution	Avg	Std
None	0.01	1.4E+05	4.2E+04
	0.001	1.9E+05	9.2E+04
25 mg	0.1	2.8E+04	2.1E+03
	0.01	3.2E+04	1.1E+04
50 mg	0.1	6.7E+03	2.2E+03
	0.01	2.1E+04	1.2E+04
100 mg	1	7.8E+02	2.2E+01
	0.1	4.9E+03	1.3E+03

P. larvae spore stock #15 was used in this experiment with a determined CFU/mL of 2.1x10^6. Meaning, after the spores were added to the glass cover slip, there should have been a CFU/mL of 2.1x10^5 after the cover slip was re-suspended in the 1 mL of sterile ddH2O. The CFU/mL that was recovered in the "no exposure" group was between 1.4x10^5 to 1.9x10^5, meaning there was between a 66.7% to 90.5%. 

Humidity inside modified anaerobic chamber

In order to determine what the humidity and temperature is like inside the modified anaerobic chamber during the ClO2 expose I set up the chambers just like I would during the actual experiment (minus the spores) and put a temperature/humidity detector inside the chamber. After six hours the temperature and humidity were:

Humidity = 96% 

Temperature = 28.8C

The temperature inside the chamber was very close to the room's temperature inside the flow hood. The humidity inside the chamber will be used to calculate the ppm of ClO2 during the exposure. I'll repeat this humidity test again using the different magnetic stir plates.

//EWW

Germinant Receptors in P. larvae - Bioinformatics

From 4-16-15.

After consulting with Dr. Bergholz, I discovered that I had not properly converted my .sra files to .fastq files. This is because all of the .sra files that I am using are paired end reads and need to be split up. Right now all my .fastq files contain both forward and reverse reads in one file and I wont be able to use it. So, I deleted all of the previously converted .fasta files and will try it again.

After consulting the sra handbook (and sra forums), I discovered I needed to use the --split-3 option to split my paired end reads when converting them to .fastq format. 

So, in the the terminal, I navigated to where my .sra files were located and after much trial and error, I used this command below:

$ /Users/Elliott/sratoolkit.2.4.5-2-mac64/bin/fastq-dump --split-3 ERR274125.sra -O /Users/Elliott/ncbi/public/fastq

This command converted my ERR274125.sra file to two .fastq files and placed it into my fastq folder. The "-O /Users..." was to tell it where to put the .fastq file once it had been generated. I chose to use ERR274125.sra as a test file and the process seemed to work. 

I then wanted to do this with all the .sra files and replaced the ERR274125.sra in the above command with just *.sra and hit enter... and it appeared to be working until I got this error: 

fastq-dump(573,0x7fff7544f310) malloc: *** error for object 0x1021e6fb0: pointer being freed was not allocated

*** set a breakpoint in malloc_error_break to debug

Abort trap: 6

Which I have no idea what it means. I will have to work on trouble shooting a bit more.

//EWW

Detection of P. larvae in Local Honey

From 3-27-15.

I attempted to amplify the 16S gene once again in the five selected P. larvae isolates from the local honey sources and the single isolate recovered from the trachea of a roach. I designed my own thermocycler parameter using the primers 8F and 16S Rev. The annealing temperature of 55.8C was used as it was between the two melting temperatures of the primers.

Thermocycler parameters:

Primer sets 8F and 16S Rev
Program name : 8F + 16S

	Temp	Time
Step 1	95.0C	2.00 m
Step 2	95.0C	1.00 m
Step 3	55.8C	1.00 m
Step 4	72.0C	1.00 m
Step 5	GoTo Step 2, repeat x 24
Step 6	72.0C	5.00 m
Step 7	20.0C	Forever

The amplicons were ran on a 1% gel at 120 volts for 30 minutes (10 uL DNA + 2 uL EZ vision loading dye). Unfortunately, the gel was blank once again (nice ladder though!). It is becoming increasingly likely that there is an issue with either my primers or the thermocycler parameter as the DNA that was used as template has been shown to be used successfully in other PCR reactions. I will re-assess this situation soon.

//EWW

Wax Worm/P. larvae LD50

From 4-17-15.

I attempted to count survival of early instar wax worms that were exposed to the bacterial endospores in the 96 well plate, however it was incredibly difficult to do so for a number of reasons. It appears that a number of the wax worms were able to escape the wells. This was evident by the discovery of several of them crawling around inside the secondary container and several wells that were mysteriously empty. It appears, that despite my attempts to keep them from escaping by flipping the plate upside down and only putting a very small hole in their covers, that they have still managed to escape.

It was also very difficult to determine if a wax worm still in the well was alive or just not moving at the moment. The wax worms were able to move underneath the oat inside their well, further obstructing my view. I would need to flip the 96 well plate back over to its correct orientation and open it up to more thoroughly assess their survival. However, that is not a feasible thing to do given the circumstances. It should be noted that it is very easy to determine if the wax worm is dead sometimes, especially when they have turned black in color - which some of them had.

Overall, here on Day 1, there was a large amount of wax worms that were unaccounted for in their wells, or were too obscured to accurately determine their survival. I will continue to monitor this plate, however my hopes are low while using this particular set-up.

New Set-up

I repeated the same method as before by transferring early instar wax worms to the wells of a 96 well plate containing a single oat soaked with spores, however this time I sealed the plate using a transparent sticky cover slip. I pieced an even smaller hole than last time in the cover over each well. I am not sure if the wax worms will still be able to escape, but it is significantly easier to observe their survival now (you can see through both the top and the bottom of the plate!).

96 well plates containing the early instar wax worms. The transparent cover slips are affixed to the plates.

The plates (x2) were incubated at 37C inside a secondary container. I will monitor their survival every 24 hours.

//EWW

Sporulation on Blood Agar

From 4-7-15.

I made more Columbian sheep's blood agar slants and plates. The agar was made by autoclaving Columbian agar base. After the agar was cool to the touch, a volume of 5% defibrinated sheep's blood was added to the molten agar. A volume of 8 mL was added to sterile glass tubes to create the slants. The tubes were tilted on their sides while cooling to create the slant (used Linda P.'s tube rack in order to achieve the tilt without dumping out the agar).

100 uL of an overnight broth culture of P. larvae 9545 (in BHI) was spread onto the agar plates. The plates were incubated, inverted, at 37C with 5% CO2 for seven days. On Saturday, April 25, I will extract the P. larvae spores from the agar.

300 uL of the overnight P. larvae 9545 broth culture in BHI was inoculated onto the agar slants. An image of how this process is performed and what it should look like is seen here. The slants were incubated at 37C in the lab incubator for ten days. On Tuesday, April 28, I will extract the P. larvae spores from the slants.

//EWW

ClO2 Study - P. larvae Spores on Glass

From 4-15-15.

The colonies have begun to form on the MYPGP plates from the last ClO2 experiment. Initial colony counts seem to show a general trend of the more ClO2 reagent the spores were exposed to, the less colonies there were. However, I will wait an additional day or two before I report official colony counts. I have found that, especially with this P. larvae bacteria, the longer I wait to count colonies the more appear and the better they come in (can take up to a week before all colonies are seen).

Below are two images to be used in comparison of the effects of ClO2 on P. larvae spores on glass:

P. larvae spores exposed to 100 mg of ClO2 reagent (dilution 10^0)

P. larvae spores exposed to no ClO2 reagent (dilution 10^0)

//EWW

Wax Worm/P. larvae LD50

From 4-4-15.

Goal: Design a new method to use in determining the LD50 of the early instar wax worms to P. larvae spores.

Background: One of the issues I was having with the previous experiments was that the wax worms were not fed a high enough concentration of spores and I never actually was able to kill them. The B. thur spores were able to cause death in the wax worms after about three days, but all the P. larvae exposed wax worms still survived (or died due to other causes). However, signs of disease was observed in some of the wax worms exposed to P. larvae by day three, but cleared up by day six. The main reason that the early instar wax worms were not fed a high enough concentration of P. larvae spores was because my titers were not very high. Fortunately, I have increased my titer of P. larvae spores through use of the Columbian sheep's blood agar. The titers have increased from ~10^3 to ~10^6.

Another issue I had ran into previously was with storing numerous wax worms together that were in the same treatment group. The main concern with this was that frequently I would count surviving wax worms after each time point and there would be one completely missing from the container. I am extremely confident that the wax worm did not escape. My conviction in this statement was cemented after several small pieces of a wax worm's head was found inside the container (missing it's body). This was observed several times and upon further investigation I discovered that the wax worms will actually eat each other! They will eat organic material, including each other if the food source is scarce, which was also true since they were only provided the BAD spiked with spores as nutrients.

Also, there was some (more than I'd like) death in the early instar wax worms at Day 1. This was often attributed to either handling issues, which became reduced once I become more experienced moving the wax worms, or drowning of the wax worms in the diet. There were a number of wax worms that would be floating in the diet at Day 1. There was only a small volume pipetted into the container (between 5-10 uL), however they are VERY small early instar wax worms.

The last big issue I had with the previous experiment was the type of containers that was being used. There was no air transfer using those containers, and even though they were opened daily to count survival, humidity was noticeably building up inside the container. I am not sure how much oxygen an early instar wax worms needs for 24 hours, but I am pretty sure with 5-10 of them inside the small tightly sealed container, there wasn't enough. Coupled with the large amount of wax worms per container (between 5 -10 depending on the experiment) and the lack of air exchange the containers themselves became very inhospitable by the end of the experiments. 

Method: For this experiment, we wished to expose the wax worms to a higher concentration of P. larvae spores (in a decreased volume), store them individually in a 96 well plate, and use a breathable cover for the plate to facilitate air exchange. The next generation of wax worms have just began to hatch from their eggs a few days ago and are now a size that is easy to work with for this experiment.

1. Add a single piece of oat meal to the bottom of a well in a 96 well plate (I am not 100% sure if they WW will eat the oatmeal, but it is meant to serve as their nutrient source over the course of this experiment.
2. Serially dilute P. larvae spore stocks in sterile ddH2O and add a volume of 3 uL directly to the top of the piece of oatmeal inside the well. Allow the liquid to be absorbed into the oatmeal (~1 minute).
3. Add an early instar wax worm is then carefully transferred to the well using a forceps. Make sure it doesn't escape the well before it is covered.
4. Cover the wells using a metal sticky cover seal. Create a very small pin sized hole in the top of the film for the wax worms to breath and allow air to be exchanged.

The smiley bug is obviously a wax worm

5. Once all the wells are covered, tip the 96 well plate upside down and place inside a secondary container. Incubate the container at 37C and check survival of the wax worms every 24 hours.

Note: the reason the plates were not covered with a more permeable (or breathable) cover is because the WW will eat that material and escape the well. This is also the reason the plates were tipped upside down- to prevent them from leaving their wells.

For this experiment,

Control:
Negative - sterile ddH2O
Positive - B. thur spores (stock conc of 10^8 CFU/mL)

Experimental treatments:
P. larvae spores- stock (2.1x10^6 CFU/mL) and dilutions: 10^-1, 10^-2, 10^-3, 10^-4
Meaning the wax worms were fed 1000 spores, 100 spores, 10 spores, 1 spore, and essentially no spores (0.1 spore).

//EWW

Germinant Receptors in P. larvae - Bioinformatics

From 4-13-15.

Downloaded Stampy & BWA from http://www.well.ox.ac.uk/software-download-registration.

From the website:

Description: Stampy is a package for the mapping of short reads from illumina sequencing machines onto a reference genome. It's recommended for most workflows, including those for genomic resequencing, RNA-Seq and Chip-seq. Stampy excels in the mapping of reads containing that contain sequence variation relative to the reference, in particular for those containing insertions or deletions. It can map reads from a highly divergent species to a reference genome for instance. Stampy achieves high sensitivity and speed by using a fast hashing algorithm and a detailed statistical model. Stampy has the following features: Maps single, paired-end and mate pair Illumina reads to a reference genome Fast: about 20 Gbase per hour in hybrid mode (using BWA) Low memory footprint: 2.7 Gb shared memory for a 3Gbase genome High sensitivity for indels and divergent reads, up to 10-15% Low mapping bias for reads with SNPs Well calibrated mapping quality scores Input: Fastq and Fasta; gzipped or plain Output: SAM, Maq's map file Optionally calculates per-base alignment posteriors Optionally processes part of the input Handles reads of up to 4500 bases The reference paper for the package can be found here. I plan to use Stampy & BWA to map all my downloaded sequences to a reference strain of P. larvae DSM 25430, which I have also downloaded the fasta file from NCBI for. //EWW

ClO2 Study - P. larvae Spores on Glass

From 4-14-15.

Repeated ClO2 gas exposure to P. larvae spores on glass cover slips as before using different weights of ClO2 reagents. The previous time this experiment was ran I used a lot of ClO2 reagents for the reactions (200 mg and 2000 mg) which resulted in a lot of spore killing as evident by the lack of colony formation on the MYPGP agar after. The 2000 mg weight actually resulted in total killing of the spores and the 200 mg almost had the same result. So, this time I will be using less ClO2 reagent.

The total dry weights of ClO2 reagents used in this experiment were 100 mg, 50 mg, 25 mg, and 0 mg. P. larvae spore stock #15 was used in this experiment.

The spores on the glass cover slips were exposed to the ClO2 gas inside the modified anaerobic chambers inside the fume hood for six hours once again.

The spores were re-suspended after and plated on MYPGP agar as before. Plates were incubated at 37C for three days.

//EWW

ClO2 Study - P. larvae Spores on Glass

From 4-11-15.

Visible P. larvae colonies have begun to form on the MYPGP agar

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


Unfortunately, there were no colony growth on the 2000 mg ClO2 weight concentration, even at the least dilute plate (10^-1). This isn't overly surprising as that concentration was relatively very high. So high in fact that it turned the plastic of the anaerobic chamber and the water inside a yellow tint. Fortunately, the yellow tint has disappeared from the container after a couple of days.
There were countable colonies in the No ClO2 and 200 mg treatment groups. Below is a graph illustrating the differences between CFU in those treatment groups:

Raw Data

Colony Counts:

	Dilution Factor
ClO2 Conc	0	-1	-2	-3	-4
None	TNTC	TNTC	9	3	0
	TNTC	TNTC	9	1	0
	TNTC	TNTC	11	9	0
	TNTC	TNTC	13	3	0
	TNTC	TNTC	9	2	0
	TNTC	TNTC	13	2	0
	TNTC	TNTC	16	3	0
	TNTC	TNTC	9	1	0
	TNTC	TNTC	5	0	0
	TNTC	TNTC	7	4	0
	TNTC	TNTC	13	2	0
	TNTC	TNTC	11	3	0
	TNTC	TNTC	9	1	0
	TNTC	TNTC	7	3	0
	TNTC	TNTC	10	0	0
200 mg	1	0	0	0	0
	2	0	0	0	0
	1	0	0	0	0
	1	0	0	0	0
	0	0	0	0	0
	3	0	0	0	0
	2	0	0	0	0
	0	0	0	0	0
	1	0	0	0	0
	1	0	0	0	0
	1	0	0	0	0
	0	0	0	0	0
	2	0	0	0	0
	0	0	0	0	0
	4	0	0	0	0

CFU/mL:
0 -1 -2 -3 -4 None N/A N/A 9.0E+04 3.0E+05 N/A N/A N/A 9.0E+04 1.0E+05 N/A N/A N/A 1.1E+05 9.0E+05 N/A N/A N/A 1.3E+05 3.0E+05 N/A N/A N/A 9.0E+04 2.0E+05 N/A N/A N/A 1.3E+05 2.0E+05 N/A N/A N/A 1.6E+05 3.0E+05 N/A N/A N/A 9.0E+04 1.0E+05 N/A N/A N/A 5.0E+04 0.0E+00 N/A N/A N/A 7.0E+04 4.0E+05 N/A N/A N/A 1.3E+05 2.0E+05 N/A N/A N/A 1.1E+05 3.0E+05 N/A N/A N/A 9.0E+04 1.0E+05 N/A N/A N/A 7.0E+04 3.0E+05 N/A N/A N/A 1.0E+05 0.0E+00 N/A 200 mg 1.0E+01 N/A N/A N/A N/A 2.0E+01 N/A N/A N/A N/A 1.0E+01 N/A N/A N/A N/A 1.0E+01 N/A N/A N/A N/A 0.0E+00 N/A N/A N/A N/A 3.0E+01 N/A N/A N/A N/A 2.0E+01 N/A N/A N/A N/A 0.0E+00 N/A N/A N/A N/A 1.0E+01 N/A N/A N/A N/A 1.0E+01 N/A N/A N/A N/A 1.0E+01 N/A N/A N/A N/A 0.0E+00 N/A N/A N/A N/A 2.0E+01 N/A N/A N/A N/A 0.0E+00 N/A N/A N/A N/A 4.0E+01 N/A N/A N/A N/A

Averages:

	0	-1	-2	-3	-4
None
Avg	N/A	N/A	1.0E+05	2.5E+05	N/A
Std	N/A	N/A	2.8E+04	2.2E+05	N/A
200 mg
Avg	1.3E+01	N/A	N/A	N/A	N/A
Std	1.2E+01	N/A	N/A	N/A	N/A

The average recovery of spores from the No ClO2 exposure group is listed above, meaning that the recovery of the P. larvae spores from the glass cover slip was between 35.7% to 89.3% (from the 2.8x10^5 CFU/mL). Which isn't too bad, but not the best either.

Graph (using the -2 data from the No ClO2 group since it had the lowest Std)

//EWW

Germinant Receptors in P. larvae - Bioinformatics

From 4-6-15.

I want to convert all the .sra files to .fastq files using the sra toolkit. I did not want to convert each of them

Fortunately, I received assistance from Oleksandr M. with the command line script to convert the formats after many hours of attempts and failures on my own.

Converting .sra to .fastq

1. In order to convert all of my .sra files to .fastq in one go, rather than type them all in individually I had to first navigate to the directory (in the command terminal) where all the executable files for the sra toolkit were located.
1. It is found in the sratoolkit.2.4.5-2-mac64 / bin
2. Once there, I entered  $ pwd    and was told that directory's location. 
1. The pwd was " /Users/Elliott/sratoolkit.2.4.5-2-mac64/bin "
3. After the source directory was identified, I navigated to the location where all the .sra files were located.
1. It is found in ncbi / public / sra
4. Once there, I entered the command to begin converting all the .sra files found in this directory to .fastq using the command $ /Users/Elliott/sratoolkit.2.4.5-2-mac64/bin/fastq-dump *.sra
1. The first part indicates the source (the sra toolkit in this case) and asterisk (*) indicates to perform that action to ALL the .sra files found in that directory. I believe I could have also told it to put all the newly converted .fastq files into a different directory, but instead it just put them in the current one with the .sra files.
2. All the .fastq files were generated into the ncbi / public / sra
5. All of the .fastq files were then manually moved from the sra directory into the newly created fastq directory located in ncbi / public/ fastq

//EWW

ClO2 Study - P. larvae Spores on Glass

Goal: Expose P. larvae spores on a glass surface to chlorine dioxide gas in order to determine the gas's effect on the spores. The glass surface will serve as somewhat of a control, as metal, plastic, and possibly wood surfaces will be implemented in the future.

Procedure:

1. A 100 uL volume of P. larvae spore stock was added to the surface of a sterile glass cover slip. The volume was allowed to evaporate for about an hour inside the laminar flow hood. 
1. For this experiment. I used Spore Stock #16 from 4-7-15, so each cover slip was inoculated with 2.8x10^5 spores.
   

   

   100 uL volume of P. larvae spore stock on glass cover slip

   
   
2. The chlorine dioxide reagents (Part A & Part B) were weighed out using he analytical balance in the Pruess lab with reagents being temporarily stored in 1.5 mL eppendorf tubes. Note: Be careful not to mix the two reagents and to change out weigh paper and scoops between each reagent.
1. For this experiment, there was only two experimental groups using chlorine dioxide- 100 mg and 1000 mg concentrations. Once the reagents are mixed their final weight concentration will be 200 mg and 2000 mg.
3. The actual ClO2 gas exposure will take place inside of the modified anaerobic chambers as shown on 10-18-15 inside of a laminar flow hood. The exposure chambers were assembled and organized also similarly as to what had been previously done. A beaker containing 50 mL of ddH2O was added to the corner of the modified anaerobic chamber to increase humidity (higher humidity increases the rate at which ClO2 is generated). A metal stir bar in combination with a magnetic stir plate were utilized in order to evenly mix the gas once it is being generated. In order to get the metal stir bar to smoothly spin inside the chamber a number of glass microscopy slides were placed on the bottom of the chamber for the stir bar to spin on. Otherwise the stir bar would be spinning on the plastic.

1. 

   Set up anaerobic chamber similarly it what it was on 11-9-14. This image does not show the sachet containing the ClO2 reagents. Note: the metal stir bar in the center (blurry spot)- it is spinning on top of several glass microscope slides that have been placed on the bottom of the chamber.

   

   Chambers set up in laminar flow hood. The ClO2 exposure will be conducted within the hood. 

   
   
4. The modified anaerobic chambers were closed and allowed to spin for an hour to build up humidity (and while I prepped everything else)



How the modified anaerobic chamber was organized. Image created using Inkscape Software.

1. After three hours of the chamber being closed and spinning the humidity was 77% and was a temperature of 26.2C.
5. The glass cover slips containing the dried P. larvae spores were aseptically transferred to the modified anaerobic chambers using sterile forceps. The cover slips (three per container) were placed inside a sterile petri plate.
6. The chlorine dioxide gas reagents were quickly and thoroughly mixed inside small sachet bags and added to the modified anaerobic chambers. The tops of the sachet bags were pinched in the opening of the chamber lid and sealed shut. The light in the hood was turned off and the opening to the hood was sealing using foam boards
1. 
   

   

   Hood sealed with foam board to protect the anaerobic chambers from reaction - ClO2 will not be generated as efficiently in the presence of light. 

   
   
7. The exposure continued for six hours inside the sealed laminar flow hood with the lights off and metal stir bar spinning.
8. After six hours, the chlorine gas concentrations was measured in each modified anaerobic chamber using chlorine gas detection tubes (previously described 10-8-14) in conjugation with the GasTec (Link) pump. A total volume of 200 mL was removed from the container.
1. For this experiment, there was significantly more chlorine gas present in the anaerobic chamber with the larger weight of chlorine dioxide gas reagent, which makes logical sense. In the container with only 200 mg the Cl detection tube only detected 75 ppm Cl. In the container with 2000 mg, there was over 500 ppm of chlorine present. The detection tube was unable to determine exactly how much Cl was present in this container since it was beyond what the detection column could detect.



Chlorine gas detection tubes from each of the experimental groups, the left is from the container with 200 mg total of reagent (100 mg each), and the right is from the container with 2000 mg total weight.

In fact, the concentration of Cl was so high in the one container that the container itself turned yellow. Even the water inside that container turned yellow. The below image shows the difference between the two containers - there is no trick with the lighting, the container on the right now has a yellow tint to it compared to the one on the left.

Notice the color differences between the two chambers that were exposed to different levels of ClO2 gas



1. Additionally, the temperature inside the hood, but not the containers, after 6 hours was 30.4C.

9. The anaerobic chamber was opened inside the laminar flow hood and the cover slips containing the P. larvae spores were aseptically transferred to a 50 mL conical tube containing 1.0 mL of sterile ddH2O. The glass cover slip was broken (shattered) using a sterile forceps while inside the tube.

10. The tube and contents were vigorously vortexed for 30 seconds and allowed to incubate at room temperature for 5 minutes.

11. The re-suspended spores were diluted in sterile ddH2O and plated on MYPGP agar. Plates were incubated at 37C for three days until colonies can be counted and spore concentration can determined.

//EWW