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