Chlorine Dioxide Pilot Study

From 1-31-15.

Counted colonies that had formed on my MYPGP agar plates from the recent chlorine dioxide exposure study. Colony counts are listed below:

		Dilutions & CFU Counts
Spore Stock	ClO2 Reagents	10^-0	10^-0	10^-0	1:2	1:2	1:2
H	None	164	152	188	80	68	62
		155	148	194	66	76	84
		190	168	156	78	72	84
		136	178	168	88	62	58
		168	166	162	74	58	66
	Avg:	162.6	162.4	173.6	77.2	67.2	70.8
	Std:	19.67	12.28	16.58	8.07	7.29	12.38
	Avg of all:			166.20			71.73
	Std of all:			16.17			9.79
H	10mg	29	34	41	14	12	9
		27	29	38	14	13	16
		23	26	45	17	9	12
		34	25	37	13	15	15
		28	32	44	15	12	12
	Avg:	28.2	29.2	41	14.6	12.2	12.8
	Std:	3.96	3.83	3.54	1.52	2.17	2.77
	Avg of all:			32.80			13.20
	Std of all:			6.96			2.31
H	100 mg	No colonies formed			No colonies formed
		Dilutions & CFU Counts
Spore Stock	ClO2 Reagents	10^-0	10^-0	10^-0	1:2	1:2	1:2
I	None	400	333	386	188	234	244
		414	376	376	222	200	220
		398	440	364	228	192	190
		380	434	358	244	182	188
		422	422	388	222	208	188
	Avg:	402.80	401.00	374.40	220.80	203.20	206.00
	Std:	16.16	45.55	13.22	20.43	19.73	25.22
	Avg of all:			392.73			210.00
	Std of all:			29.97			21.82
I	10 mg	16	12	10	7	8	7
		12	16	17	4	8	5
		11	15	13	5	9	10
		19	20	13	7	6	11
		20	21	16	8	6	9
	Avg:	15.60	16.80	13.80	6.20	7.40	8.40
	Std:	4.04	3.70	2.77	1.64	1.34	2.41
	Avg of all:			15.40			7.33
	Std of all:			3.52			1.95
I	100 mg	No colonies formed			No colonies formed

Calculating CFU:
Spore Stock H = 84,000 CFU/mL (8.4x10^4)
Spore Stock I = 32,000 CFU/mL (3.2x10^4)
---------------------------> pelleted & re-suspended in 500 uL ddH2O
Spore Stock H = 1.68x10^5 CFU/mL
Spore Stock I = 6.4x10^4 CFU/mL
---------------------------> 50 uL of spore stocks added to glass cover slip
Cover slips exposed to ClO2 gas
---------------------------> re-suspend cover slip spores in 1000 uL of ddH2O
---------------------------> 10 uL volumes plated onto MYPGP agar

Equation used to calculate concentration of Spore Stocks:

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

For Spore Stock H:
           32.8/166.2 CFU x 100% = 19.7% survival of spores
                        Meaning that the 10 mg concentration of ClO2 reagent was ~80% effective at killing   the spores

For Spore Stock I
         15.4/392.73 CFU x 100% = 3.92% survival of spores
                     Meaning that the 10 mg concentration of ClO2 reagent was ~96% effective at killing the spores

Unfortunately, my original spores stocks were at a relatively low concentration to begin with (around 10^4 CFU), and this limited what we could see at the 100 mg concentration since we could only see about a 3 log killing rate and nothing more. Realistically, the spore stocks should be around 10^8 or 10^9, so I will have to work on improving my ability to recover and purify P. larvae spores.


/EWW