Dictyostelium Predation Assay for transposon mutagensis of pDR1 Part(3)

Date: Oct./28/2014
The protocol in the link
Plate name : individual colonies(contain 10 colonies), pDR1/kana. transposon mutagensis.
 The result: after 4 days incubation at room temp. in a dark place

Plaques in Kleb. plate=32 plaques in dilution 1/1000 (-3)

Plaque forming unit pfu =# plaque X dilution factor/volume of inoculum
                                       32 X10^3/10 = 3.2 X10^3
2 μl of  8 Dicty. dilutions that contain(6.4 X 10^3, 6.4 X 10^2, 6.4 X 10 ,6.4 , 0.64,....) Dicty. cells were used on sm2 agar in four replicate for each dilution.
The result after 4 days :
Controls: 

• pDR1plague :(0,-1) 
•  cosmid control.plague(0,-1,-2,-3,-4)
• Clones shows sensitivity to Dictyostelium predation:

plague (0,-1,-2,-3,-4) 
8-A5 named: pDR1tn8

Dictyostelium Predation Assay for transposon mutagensis of pDR1 Part(2)

Date: Oct./25/2014
The protocol in the link
Plate 4, pDR1/kana. transposon mutagensis.
 The result: after 4 days incubation at room temp. in a dark place

Plaques in Kleb. plate=23 plaques in dilution 1/1000 (-3)

Plaque forming unit pfu =# plaque X dilution factor/volume of inoculum
                                       23 X10^3/10 = 2.3 X10^3
2 μl of  8 Dicty. dilutions that contain(4.6 X 10^3, 4.6 X 10^2,4.6 X 10 ,4.6 , 0.46 ,....) Dicty. cells were used on sm2 agar in four replicate for each dilution.
The result after 4 days :
Controls: 

• pDR1plague :(0,-1,-2) 
•  cosmid control.plague(0,-1,-2,-3,-4)
• Clones shows sensitivity to Dictyostelium predation:

plague (0,-1,-2,-3) 
6-A7 named: pDR1tn6 - small palques-

plague(0,-1,-2,-3,-4)
7-A6   named:   pDR1tn6
The result after 5 days incubation look like same 

Plaques measurement by Dicty. predation assay

Date oct/16/2014
Dicty preparation and dilution as link

The result

In Kleb. PFU= (average # of plaques x d.f)/volume of culture

 =6 X 10⁵ /10   =  6.0 X 10⁴

3 different dilution of  Dicty. in sterile distal water( 0, 1/100, 1/10000) were used  on different clones and strains of bacteria. Each clone or strain was inoculated on sm2 agar 

2  μl that contain dose of (1.6 X10^3, 1.6 X 10^2 , 1.6 X 10 , 1.6 , 0.16 ) of each Dicty. dilution titration containing(3.0 X 10⁵ ,3.0 X10³ and 3.0 X 10) was dropped on sm2 agar that have been inoculated previously with the specific bacterial clone, and plate were kept at room temperature in a dark place.

After 3 days incubation, the diameter of plaque in mm

Bacterial strain		0 Dicty. dilution	1:100 dilution	1:10000 dilution
DH5 alfa E.coli	11		5	-
EPI 100 E.coli	10.5		5	-
Klebsiella.	13		5	-
pDR Cosmid control	11		5	-
pDR1	3.5		3	-
pDR2	3		-	-
pDR4	5		-	-
pDR5	3		-	-
pDR6	-		-	-
Stenotrophomonas maltophilia BAA2423(K279a)	5.5		-	-
S.m BAA84(D457)	5		-	-
S.m M	5		-	-
S.m N	-		-	-
S.m H	4		-	-

After 4 days incubation, the diameter of plaque in mm

Bacterial strain		0 Dicty. dilution	1:100 dilution	1:10000 dilution
DH5 alfa E.coli	18		12	-
EPI 100 E.coli	19		8	8
Klebsiella.	30		16	10
pDR Cosmid control	18		12	9
pDR1	14		9	-
pDR2	5		-	-
pDR4	8		-	-
pDR5	7		-	-
pDR6	9		-	-
Stenotrophomonas maltophilia BAA2423(K279a)	9		-	-
S.m BAA84(D457)	7		-	-
S.m M	7		-	-
S.m N	3		-	-
S.m H	7		-	-

After 5 days incubation, the diameter of plaque in mm

Bacterial strain		0 Dicty. dilution	1:100 dilution	1:10000 dilution
DH5 alfa E.coli	18		17	8
EPI 100 E.coli	30		10	13
Klebsiella.	40		25	17
pDR Cosmid control	25		17	11
pDR1	14		10	5
pDR2	5		-	-
pDR4	8		-	-
pDR5	9		-	-
pDR6	10		-	-
Stenotrophomonas maltophilia BAA2423(K279a)	18		8	-
S.m BAA84(D457)	7		-	-
S.m M	9		-	-
S.m N	3		-	-
S.m H	9		-	-