Hyper Stacks 1994 May

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MacBinary | 1993-09-23 | 50.9 KB | [STAK/WILD]
open in:
MacOS 8.1 extracted |
Win98 extracted |
DOS extracted
view JSON data |
view as text

This file was processed as: MacBinary (archive/macBinary).

You can browse this item here: Protocol Book
Confidence	Program	Detection	Match Type	Support
`100%`	dexvert	HyperCard Stack `(archive/hypercard)`	idMeta	Supported
`10%`	dexvert	MacBinary `(archive/macBinary)`	fallback	Supported
`100%`	file	MacBinary II, inited, Tue May 25 09:05:18 1993, modified Thu Sep 23 13:14:22 1993, creator HyperCard, type HyperCard stack, 44192 bytes "Protocol Book" , at 0xad20 7559 bytes resource	default (weak)
`99%`	file	data	default
`74%`	TrID	Mac HyperCard stack (MacBinary)	default
`25%`	TrID	MacBinary 2	default (weak)
`100%`	lsar	MacBinary	default
id metadata
key	value
macFileType	`[STAK]`
macFileCreator	`[WILD]`
hex view
+--------+-------------------------+-------------------------+--------+--------+
|00000000| 00 0d 50 72 6f 74 6f 63 | 6f 6c 20 42 6f 6f 6b 00 |..Protoc|ol Book.|
|00000010| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000020| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000030| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000040| 00 53 54 41 4b 57 49 4c | 44 01 00 00 00 00 00 00 |.STAKWIL|D.......|
|00000050| 00 00 00 00 00 ac a0 00 | 00 1d 87 a8 27 ca 0e a8 |........|....'...|
|00000060| c7 89 ee 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000070| 00 00 00 00 00 00 00 00 | 00 00 81 81 a7 e8 00 00 |........|........|
|00000080| 00 00 08 00 53 54 41 4b | ff ff ff ff 00 00 00 00 |....STAK|........|
|00000090| 00 00 00 0a 00 00 ac a0 | 00 00 08 00 00 00 00 00 |........|........|
|000000a0| 00 00 00 00 00 00 00 01 | 00 00 0a ab 00 00 00 07 |........|........|
|000000b0| 00 00 09 c0 00 00 08 3a | 00 00 00 00 00 00 00 00 |.......:|........|
|000000c0| 00 00 0f 32 00 00 00 00 | 00 03 00 00 5c 00 00 00 |...2....|....\...|
|000000d0| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|000000e0| 02 10 80 00 02 10 80 00 | 02 10 80 00 02 10 80 00 |........|........|
|000000f0| 91 ec 54 1e 00 00 00 01 | 00 49 00 50 01 9f 02 50 |..T.....|.I.P...P|
|00000100| 00 00 00 00 01 e0 02 80 | 00 00 00 00 00 00 00 00 |........|........|
|00000110| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000120| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000130| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000140| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000150| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000160| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000170| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000180| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000190| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|000001a0| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|000001b0| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|000001c0| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|000001d0| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|000001e0| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|000001f0| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000200| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000210| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000220| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000230| 00 00 0d 39 00 00 0c ef | 01 56 02 00 00 00 00 00 |...9....|.V......|
|00000240| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000250| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000260| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000270| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000280| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000290| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|000002a0| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|000002b0| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|000002c0| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|000002d0| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|000002e0| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|000002f0| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000300| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000310| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000320| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000330| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000340| 00 00 00 00 00 00 00 00 | 80 00 00 00 08 00 00 00 |........|........|
|00000350| 88 00 22 00 88 00 22 00 | 88 88 22 22 88 88 22 22 |.."...".|..""..""|
|00000360| 88 aa 22 aa 88 aa 22 aa | cc aa 33 aa cc aa 33 aa |.."...".|..3...3.|
|00000370| ee aa bb aa ee aa bb aa | ee bb bb ee ee bb bb ee |........|........|
|00000380| ff bb ff ee ff bb ff ee | ff bb ff ff ff bb ff ff |........|........|
|00000390| 80 10 02 20 01 08 40 04 | ff ff ff ff ff ff ff ff |... ..@.|........|
|000003a0| 88 22 88 22 88 22 88 22 | 11 22 44 88 11 22 44 88 |."."."."|."D.."D.|
|000003b0| c4 80 0c 68 43 02 30 26 | b1 30 03 1b d8 c0 0c 8d |...hC.0&|.0......|
|000003c0| aa 00 aa 00 aa 00 aa 00 | 88 22 55 22 88 22 55 22 |........|."U"."U"|
|000003d0| 88 55 22 55 88 55 22 55 | 77 dd 77 dd 77 dd 77 dd |.U"U.U"U|w.w.w.w.|
|000003e0| 80 00 00 00 00 00 00 00 | aa 55 aa 55 aa 55 aa 55 |........|.U.U.U.U|
|000003f0| 03 84 48 30 0c 02 01 01 | 82 44 39 44 82 01 01 01 |..H0....|.D9D....|
|00000400| 88 14 22 41 88 41 22 14 | 80 80 41 3e 08 08 14 e3 |.."A.A".|..A>....|
|00000410| 22 04 8c 74 22 17 98 10 | be 80 88 08 eb 08 88 80 |"..t"...|........|
|00000420| 25 c8 32 89 64 24 4c 92 | a2 9c 41 be 2a c9 14 eb |%.2.d$L.|..A.*...|
|00000430| 40 a0 00 00 04 0a 00 00 | 80 40 20 00 02 04 08 00 |@.......|.@ .....|
|00000440| aa 00 80 00 88 00 80 00 | ff 80 80 80 80 80 80 80 |........|........|
|00000450| 08 1c 22 c1 80 01 02 04 | ff 80 80 80 ff 08 08 08 |..".....|........|
|00000460| f8 74 22 47 8f 17 22 71 | bf 00 bf bf b0 b0 b0 b0 |.t"G.."q|........|
|00000470| ff 7f be 5d a2 41 80 00 | fa f5 fa f5 a0 50 a0 50 |...].A..|.....P.P|
|00000480| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000490| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|000004a0| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|000004b0| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|000004c0| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|000004d0| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|000004e0| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|000004f0| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000500| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000510| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000520| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000530| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000540| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000550| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000560| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000570| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000580| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000590| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|000005a0| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|000005b0| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|000005c0| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|000005d0| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|000005e0| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|000005f0| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000600| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000610| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000620| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000630| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000640| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000650| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000660| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000670| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000680| 6f 6e 20 6f 70 65 6e 53 | 74 61 63 6b 0d 67 6c 6f |on openS|tack.glo|
|00000690| 62 61 6c 20 69 6e 43 6f | 6c 6f 72 0d 73 74 61 72 |bal inCo|lor.star|
|000006a0| 74 20 75 73 69 6e 67 20 | 73 74 61 63 6b 20 22 4c |t using |stack "L|
|000006b0| 61 62 48 65 6c 70 65 72 | 22 0d 69 66 20 69 6e 43 |abHelper|".if inC|
|000006c0| 6f 6c 6f 72 20 69 73 20 | 65 6d 70 74 79 20 74 68 |olor is |empty th|
|000006d0| 65 6e 20 70 75 74 20 74 | 72 75 65 20 69 6e 74 6f |en put t|rue into|
|000006e0| 20 69 6e 43 6f 6c 6f 72 | 0d 63 48 43 73 65 74 55 | inColor|.cHCsetU|
|000006f0| 70 20 69 6e 43 6f 6c 6f | 72 2c 20 46 61 6c 73 65 |p inColo|r, False|
|00000700| 2c 20 22 48 43 63 6f 6c | 6f 72 69 7a 65 22 0d 48 |, "HCcol|orize".H|
|00000710| 43 63 6f 6c 6f 72 69 7a | 65 0d 70 61 73 73 20 6f |Ccoloriz|e.pass o|
|00000720| 70 65 6e 53 74 61 63 6b | 0d 65 6e 64 20 6f 70 65 |penStack|.end ope|
|00000730| 6e 53 74 61 63 6b 0d 00 | 2c 20 46 61 6c 73 65 2c |nStack..|, False,|
|00000740| 20 22 48 43 63 6f 6c 6f | 72 69 7a 65 22 0d 48 43 | "HCcolo|rize".HC|
|00000750| 63 6f 6c 6f 72 69 7a 65 | 0d 65 6e 64 20 6f 70 65 |colorize|.end ope|
|00000760| 6e 53 74 61 63 6b 00 74 | 61 72 74 20 75 73 69 6e |nStack.t|art usin|
|00000770| 67 20 73 74 61 63 6b 20 | 22 52 65 63 69 70 65 20 |g stack |"Recipe |
|00000780| 42 6f 6f 6b 22 0d 73 74 | 61 72 74 20 75 73 69 6e |Book".st|art usin|
|00000790| 67 20 73 74 61 63 6b 20 | 22 4c 61 62 50 72 65 70 |g stack |"LabPrep|
|000007a0| 22 0d 73 74 61 72 74 20 | 75 73 69 6e 67 20 73 74 |".start |using st|
|000007b0| 61 63 6b 20 22 4d 65 74 | 68 22 0d 73 74 61 72 74 |ack "Met|h".start|
|000007c0| 20 75 73 69 6e 67 20 73 | 74 61 63 6b 20 22 54 69 | using s|tack "Ti|
|000007d0| 6d 65 20 26 20 44 61 74 | 65 22 0d 73 74 61 72 74 |me & Dat|e".start|
|000007e0| 20 75 73 69 6e 67 20 73 | 74 61 63 6b 20 22 4e 6f | using s|tack "No|
|000007f0| 74 65 73 22 0d 73 74 61 | 72 74 20 75 73 69 6e 67 |tes".sta|rt using|
|00000800| 20 73 74 61 63 6b 20 22 | 50 65 72 69 6f 64 69 63 | stack "|Periodic|
|00000810| 20 54 61 62 6c 65 22 0d | 63 72 65 61 74 65 4d 65 | Table".|createMe|
|00000820| 6e 75 0d 63 48 43 73 65 | 74 55 70 20 54 72 75 65 |nu.cHCse|tUp True|
|00000830| 2c 20 46 61 6c 73 65 2c | 20 22 48 43 63 6f 6c 6f |, False,| "HCcolo|
|00000840| 72 69 7a 65 22 0d 48 43 | 63 6f 6c 6f 72 69 7a 65 |rize".HC|colorize|
|00000850| 0d 65 6e 64 20 6f 70 65 | 6e 53 74 61 63 6b 00 6f |.end ope|nStack.o|
|00000860| 22 20 6f 72 20 22 4f 4b | 22 0d 69 66 20 69 74 3d |" or "OK|".if it=|
|00000870| 22 4f 4b 22 20 74 68 65 | 6e 0d 64 6f 4d 65 6e 75 |"OK" the|n.doMenu|
|00000880| 00 00 02 00 4d 41 53 54 | ff ff ff ff 00 00 00 00 |....MAST|........|
|00000890| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|000008a0| 00 00 50 3a 00 01 f9 c0 | 00 00 94 ab 00 00 54 b6 |..P:....|......T.|
|000008b0| 00 05 4c ef 00 05 52 39 | 00 01 f7 74 00 05 55 32 |..L...R9|...t..U2|
|000008c0| 00 05 5f 69 00 03 9b 95 | 00 00 00 00 00 05 1e 95 |.._i....|........|
|000008d0| 00 04 c9 d0 00 00 00 00 | 00 02 e1 2a 00 00 00 00 |........|...*....|
|000008e0| 00 02 6f d2 00 00 00 00 | 00 00 00 00 00 04 8d a9 |..o.....|........|
|000008f0| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000900| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000910| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000920| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000930| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000940| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000950| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000960| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000970| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000980| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000990| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|000009a0| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|000009b0| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|000009c0| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|000009d0| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|000009e0| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|000009f0| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000a00| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000a10| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000a20| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000a30| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000a40| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000a50| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000a60| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000a70| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00000a80| 00 00 00 80 4c 49 53 54 | 00 00 08 3a 00 00 00 00 |....LIST|...:....|
|00000a90| 00 00 00 01 00 00 08 00 | 00 00 00 07 00 84 00 02 |........|........|
|00000aa0| 00 20 00 03 c0 00 01 7d | 00 00 00 07 00 00 00 00 |. .....}|........|
|00000ab0| 00 00 0b b6 00 07 00 00 | 00 00 00 00 00 00 00 00 |........|........|
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|00000b50| 20 03 fd 52 a0 1e 9c 46 | 48 56 4d b9 01 4e bc 78 | ..R...F|HVM..N.x|
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|00000b70| c7 1b c0 ad 94 84 e6 bf | 95 ee 57 24 b1 b1 ef a3 |........|..W$....|
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|00000b90| 01 ae 5b 28 66 c1 12 a8 | 51 6b 1e 68 00 00 18 d2 |..[(f...|Qk.h....|
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|00000be0| d7 7e ee ef 77 fe ff db | c7 e5 ff 63 bf ee ba d7 |.~..w...|...c....|
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|00000c00| bd fe 6f 33 86 5a 9e 3f | d5 f5 ef 79 1d 27 cd ff |..o3.Z.?|...y.'..|
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|00000c40| ff fd 7d ff fe fd 77 c2 | fe ff fb d7 ee b7 6e ff |..}...w.|......n.|
|00000c50| fb ef ff 7d fe f7 d7 ff | 2f e7 3e f5 6b 6f bd ff |...}....|/.>.ko..|
|00000c60| 7a d7 cf fd f3 7e fc 6e | f3 fd 4d 5f ff b3 67 cf |z....~.n|..M_..g.|
|00000c70| bf ba bd 1e dd 59 ff 7d | f5 bf 06 af fc 3f ee 3e |.....Y.}|.....?.>|
|00000c80| df ef fb ee 7f bd bf bc | 32 7e b4 7f d7 f2 fd f3 |........|2~......|
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|00000ca0| 77 f7 77 78 00 00 11 95 | a0 7b 5f ff d7 de eb fb |w.wx....|.{_.....|
|00000cb0| eb e3 bf ff f3 ff ff bf | df 77 fd bf fa 6f df fb |........|.w...o..|
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|00000cd0| ff 7f fe fd fa 3e ff 7b | ef 7f df f3 f7 f9 ff d7 |.....>.{|........|
|00000ce0| ff fd fd ef 7f ff df ff | f3 7d ff ff f7 bf b7 d7 |........|.}......|
|00000cf0| 7f ae ff 7f ff ff bf df | fb dd f7 ff bf ff 6e ef |........|......n.|
|00000d00| cf bf 7e 3f df ff 7f 8f | ff b7 ff ff 7f ee ff 5f |..~?....|......._|
|00000d10| 7f fe f9 fd ff 3f ff fa | 85 fb f5 ff fd 7b ff ff |.....?..|.....{..|
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|00000d40| 04 29 4d 01 8e b2 88 d7 | ea e5 a0 34 fc a1 02 34 |.)M.....|...4...4|
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|00000d60| 62 38 0d 01 b3 2d a9 c8 | 6a 5c cd 15 02 6e e0 c1 |b8...-..|j\...n..|
|00000d70| 15 78 10 0f 54 80 73 06 | 17 db d8 8a 00 01 c5 c9 |.x..T.s.|........|
|00000d80| 8c 74 84 27 50 0e 06 01 | 3d 48 e9 00 86 ad 32 e8 |.t.'P...|=H....2.|
|00000d90| 40 47 ad 05 06 80 92 d1 | ca 24 dd a0 05 10 0c 76 |@G......|.$.....v|
|00000da0| 00 46 12 18 a0 e4 80 50 | 1c e1 05 34 00 00 14 d0 |.F.....P|...4....|
|00000db0| a0 53 53 6f 3c 42 ea 48 | 2b ab 9f 3f 7d 30 5c 14 |.SSo<B.H|+..?}0\.|
|00000dc0| 4e 7a 19 e0 fc 15 bd 03 | 9d f0 85 bd ef 67 fd b6 |Nz......|.....g..|
|00000dd0| df c5 06 72 3b 3f ec d4 | 6f 1d fe ed eb cc f4 d1 |...r;?..|o.......|
|00000de0| ee af d6 22 26 92 8d 7a | 61 ae a8 6e 4d 5f dd 44 |..."&..z|a..nM_.D|
|00000df0| a2 6e ff 4e a5 ba 17 97 | 1d 92 cf 15 ba da b9 86 |.n.N....|........|
|00000e00| 18 d9 c1 f1 8d 9f 80 f7 | 82 8e f6 74 df ed 61 07 |........|...t..a.|
|00000e10| b2 fd af f0 a5 4e c4 7d | 57 b8 d4 41 0f 03 7d f1 |.....N.}|W..A..}.|
|00000e20| 27 68 58 5b 70 7b db 43 | 93 e6 86 81 bb 45 2d 30 |'hX[p{.C|.....E-0|
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|00005360| 6d 62 72 61 6e 65 20 70 | 65 72 6d 65 61 62 69 6c |mbrane p|ermeabil|
|00005370| 69 7a 61 74 69 6f 6e 20 | 6c 65 61 64 73 20 74 6f |ization |leads to|
|00005380| 20 74 68 65 20 75 70 74 | 61 6b 65 20 6f 66 20 6d | the upt|ake of m|
|00005390| 61 63 72 6f 6d 6f 6c 65 | 63 75 6c 65 73 2e 20 45 |acromole|cules. E|
|000053a0| 6e 6c 69 67 68 74 65 6e | 69 6e 67 20 64 61 74 61 |nlighten|ing data|
|000053b0| 20 6f 6e 20 74 68 65 20 | 70 72 6f 63 65 73 73 20 | on the |process |
|000053c0| 61 72 65 20 73 70 61 72 | 73 65 2c 20 61 6e 64 20 |are spar|se, and |
|000053d0| 77 65 20 61 72 65 20 66 | 61 72 20 66 72 6f 6d 20 |we are f|ar from |
|000053e0| 75 6e 64 65 72 73 74 61 | 6e 64 69 6e 67 20 68 6f |understa|nding ho|
|000053f0| 77 20 69 74 20 61 63 74 | 75 61 6c 6c 79 20 6f 63 |w it act|ually oc|
|00005400| 63 75 72 73 2e 0d 28 46 | 6f 63 75 73 20 31 32 20 |curs..(F|ocus 12 |
|00005410| 4e 75 6d 62 65 72 20 32 | 20 70 33 38 2d 34 30 2c |Number 2| p38-40,|
|00005420| 20 47 65 6e 65 74 69 63 | 20 45 6e 67 69 6e 65 65 | Genetic| Enginee|
|00005430| 72 69 6e 67 2c 20 56 6f | 6c 2e 20 31 32 2c 20 70 |ring, Vo|l. 12, p|
|00005440| 32 37 35 2d 32 39 35 29 | 0d 2d 2d 2d 2d 2d 2d 2d |275-295)|.-------|
|00005450| 2d 2d 2d 0d 4d 61 74 65 | 72 69 61 6c 73 3a 0d 0d |---.Mate|rials:..|
|00005460| 20 20 20 20 20 20 20 20 | 20 20 20 20 20 20 20 20 |        |        |
|00005470| 20 20 20 20 20 20 20 20 | 20 20 20 20 20 20 20 20 |        |        |
|00005480| 20 20 20 20 20 20 20 20 | 20 20 20 20 20 20 20 20 |        |        |
|00005490| 20 20 53 74 65 72 69 6c | 69 7a 61 74 69 6f 6e 0d |  Steril|ization.|
|000054a0| 53 4f 42 20 6d 65 64 69 | 75 6d 3a 0d 43 68 65 6d |SOB medi|um:.Chem|
|000054b0| 69 63 61 6c 20 20 20 20 | 20 20 20 20 20 20 20 20 |ical    |        |
|000054c0| 20 20 20 20 20 20 20 20 | 20 20 20 20 20 67 6d 20 |        |     gm |
|000054d0| 70 65 72 20 6c 69 74 65 | 72 20 20 20 20 20 61 75 |per lite|r     au|
|000054e0| 74 6f 63 6c 61 76 65 64 | 0d 62 61 63 74 6f 2d 74 |toclaved|.bacto-t|
|000054f0| 72 79 70 74 6f 6e 65 20 | 20 20 20 20 20 20 20 20 |ryptone |        |
|00005500| 20 20 20 20 20 20 20 20 | 20 20 20 20 20 20 20 32 |        |       2|
|00005510| 30 20 20 20 20 20 20 20 | 20 20 20 20 40 31 32 31 |0       |    @121|
|00005520| 20 a1 43 20 31 35 20 6d | 69 6e 2e 0d 62 61 63 74 | .C 15 m|in..bact|
|00005530| 6f 2d 79 65 61 73 74 2d | 65 78 74 72 61 63 74 20 |o-yeast-|extract |
|00005540| 20 20 20 20 20 20 20 20 | 20 20 20 20 20 20 20 20 |        |        |
|00005550| 20 20 20 35 0d 4e 61 43 | 6c 20 20 20 20 20 20 20 |   5.NaC|l       |
|00005560| 20 20 20 20 20 20 20 20 | 20 20 20 20 20 20 20 20 |        |        |
|00005570| 20 20 20 20 20 20 20 20 | 20 20 20 20 30 2e 35 38 |        |    0.58|
|00005580| 34 0d 4b 43 6c 20 20 20 | 20 20 20 20 20 20 20 20 |4.KCl   |        |
|00005590| 20 20 20 20 20 20 20 20 | 20 20 20 20 20 20 20 20 |        |        |
|000055a0| 20 20 20 20 20 20 20 20 | 20 30 2e 31 38 36 0d 0d |        | 0.186..|
|000055b0| 57 42 20 6d 65 64 69 75 | 6d 3a 20 20 20 20 20 20 |WB mediu|m:      |
|000055c0| 20 20 20 20 20 20 20 20 | 20 20 20 20 20 20 20 20 |        |        |
|000055d0| 20 20 20 20 20 20 20 20 | 20 20 20 20 20 20 20 20 |        |        |
|000055e0| 20 61 75 74 6f 63 6c 61 | 76 65 64 0d 31 30 25 20 | autocla|ved.10% |
|000055f0| 28 76 2f 76 29 20 67 6c | 79 63 65 72 6f 6c 20 69 |(v/v) gl|ycerol i|
|00005600| 6e 20 77 61 74 65 72 20 | 20 20 20 20 20 20 20 20 |n water |        |
|00005610| 20 20 20 20 20 20 20 20 | 20 20 20 20 20 20 20 40 |        |       @|
|00005620| 31 32 31 20 a1 43 20 31 | 35 20 6d 69 6e 2e 0d 0d |121 .C 1|5 min...|
|00005630| 32 20 4d 20 4d 67 b1 b1 | 20 73 74 6f 63 6b 3a 0d |2 M Mg..| stock:.|
|00005640| 43 68 65 6d 69 63 61 6c | 20 20 20 20 20 20 20 20 |Chemical|        |
|00005650| 20 20 20 20 20 20 20 20 | 20 20 20 20 20 20 20 20 |        |        |
|00005660| 20 20 67 6d 20 70 65 72 | 20 31 30 30 20 6d 6c 20 |  gm per| 100 ml |
|00005670| 20 20 20 61 75 74 6f 63 | 6c 61 76 65 64 0d 4d 67 |   autoc|laved.Mg|
|00005680| 43 6c aa b3 36 48 aa 4f | 20 20 20 20 20 20 20 20 |Cl..6H.O|        |
|00005690| 20 20 20 20 20 20 20 20 | 20 20 20 20 20 20 20 20 |        |        |
|000056a0| 20 20 20 20 32 30 2e 33 | 33 20 20 20 20 20 20 20 |    20.3|3       |
|000056b0| 20 40 31 32 31 20 a1 43 | 20 31 35 20 6d 69 6e 2e | @121 .C| 15 min.|
|000056c0| 0d 4d 67 53 4f a2 b3 37 | 48 aa 4f 20 20 20 20 20 |.MgSO..7|H.O     |
|000056d0| 20 20 20 20 20 20 20 20 | 20 20 20 20 20 20 20 20 |        |        |
|000056e0| 20 20 20 20 20 20 20 32 | 34 2e 36 35 0d 0d 32 20 |       2|4.65..2 |
|000056f0| 4d 20 67 6c 75 63 6f 73 | 65 3a 0d 43 68 65 6d 69 |M glucos|e:.Chemi|
|00005700| 63 61 6c 20 20 20 20 20 | 20 20 20 20 20 20 20 20 |cal     |        |
|00005710| 20 20 20 20 20 20 20 20 | 20 20 20 20 20 67 6d 20 |        |     gm |
|00005720| 70 65 72 20 31 30 30 20 | 6d 6c 20 20 20 20 66 69 |per 100 |ml    fi|
|00005730| 6c 74 72 61 74 69 6f 6e | 0d 44 2d 67 6c 75 63 6f |ltration|.D-gluco|
|00005740| 73 65 20 20 20 20 20 20 | 20 20 20 20 20 20 20 20 |se      |        |
|00005750| 20 20 20 20 20 20 20 20 | 20 20 20 20 20 20 20 33 |        |       3|
|00005760| 36 2e 30 34 0d 0d 53 4f | 43 20 6d 65 64 69 75 6d |6.04..SO|C medium|
|00005770| 3a 0d 39 38 20 6d 6c 20 | 53 4f 42 20 70 6c 75 73 |:.98 ml |SOB plus|
|00005780| 20 31 20 6d 6c 20 73 74 | 65 72 69 6c 65 20 32 20 | 1 ml st|erile 2 |
|00005790| 4d 20 4d 67 b1 b1 20 73 | 74 6f 63 6b 20 61 6e 64 |M Mg.. s|tock and|
|000057a0| 20 31 20 6d 6c 20 32 20 | 4d 20 67 6c 75 63 6f 73 | 1 ml 2 |M glucos|
|000057b0| 65 20 73 74 6f 63 6b 2e | 0d 0d 2d 2d 2d 2d 2d 2d |e stock.|..------|
|000057c0| 2d 2d 2d 2d 0d 4d 65 74 | 68 6f 64 73 3a 0d 0d 50 |----.Met|hods:..P|
|000057d0| 72 65 70 61 72 61 74 69 | 6f 6e 20 6f 66 20 45 6c |reparati|on of El|
|000057e0| 65 63 74 72 6f 2d 43 6f | 6d 70 65 74 65 6e 74 20 |ectro-Co|mpetent |
|000057f0| 43 65 6c 6c 73 3a 0d 20 | 20 20 20 20 4f 6e 65 20 |Cells:. |    One |
|00005800| 6c 6f 6f 70 66 75 6c 20 | 6f 66 20 43 52 45 37 20 |loopful |of CRE7 |
|00005810| 77 61 73 20 75 73 65 64 | 20 74 6f 20 69 6e 6f 63 |was used| to inoc|
|00005820| 75 6c 61 74 65 20 35 20 | 6d 6c 20 6f 66 20 53 4f |ulate 5 |ml of SO|
|00005830| 42 20 61 6e 64 20 73 68 | 61 6b 65 64 20 61 74 20 |B and sh|aked at |
|00005840| 72 6f 6f 6d 20 74 65 6d | 70 2e 20 66 6f 72 20 32 |room tem|p. for 2|
|00005850| 34 20 68 72 2e 20 54 68 | 69 73 20 77 61 73 20 74 |4 hr. Th|is was t|
|00005860| 68 65 6e 20 75 73 65 64 | 20 61 73 20 69 6e 6f 63 |hen used| as inoc|
|00005870| 75 6c 75 6d 20 74 6f 20 | 69 6e 6f 63 75 6c 61 74 |ulum to |inoculat|
|00005880| 65 20 31 20 6c 69 74 65 | 72 20 6f 66 20 53 4f 42 |e 1 lite|r of SOB|
|00005890| 20 74 69 6c 6c 20 4f 44 | a4 bc bc 20 72 65 61 63 | till OD|... reac|
|000058a0| 68 65 64 20 30 2e 36 2e | 0d 20 20 20 20 20 43 65 |hed 0.6.|.     Ce|
|000058b0| 6c 6c 73 20 77 65 72 65 | 20 74 68 65 6e 20 68 61 |lls were| then ha|
|000058c0| 72 76 65 73 74 65 64 20 | 28 33 30 30 30 20 67 2c |rvested |(3000 g,|
|000058d0| 20 31 35 20 6d 69 6e 2e | 29 20 69 6e 20 34 2a 32 | 15 min.|) in 4*2|
|000058e0| 35 30 20 6d 6c 20 62 6f | 74 74 6c 65 73 20 61 6e |50 ml bo|ttles an|
|000058f0| 64 20 77 61 73 68 65 64 | 20 74 77 69 63 65 20 77 |d washed| twice w|
|00005900| 69 74 68 20 31 32 35 20 | 6d 6c 20 57 42 20 61 6e |ith 125 |ml WB an|
|00005910| 64 20 63 6f 6d 62 69 6e | 65 64 20 69 6e 74 6f 20 |d combin|ed into |
|00005920| 32 20 62 6f 74 74 6c 65 | 73 20 61 66 74 65 72 20 |2 bottle|s after |
|00005930| 73 65 63 6f 6e 64 20 77 | 61 73 68 2e 20 54 68 65 |second w|ash. The|
|00005940| 20 73 75 70 65 72 6e 61 | 74 61 6e 74 20 77 61 73 | superna|tant was|
|00005950| 20 70 6f 75 72 65 64 20 | 6f 66 66 20 61 6e 64 20 | poured |off and |
|00005960| 63 65 6c 6c 20 77 61 73 | 20 72 65 73 75 73 70 65 |cell was| resuspe|
|00005970| 6e 64 20 69 6e 20 74 68 | 65 20 72 65 73 69 64 75 |nd in th|e residu|
|00005980| 61 6c 20 73 75 70 65 72 | 6e 61 74 61 6e 74 2c 20 |al super|natant, |
|00005990| 7e 20 32 20 6d 6c 20 69 | 6e 20 65 61 63 68 20 62 |~ 2 ml i|n each b|
|000059a0| 6f 74 74 6c 65 2e 20 53 | 74 6f 72 65 64 20 61 73 |ottle. S|tored as|
|000059b0| 20 32 30 30 20 b5 6c 20 | 61 6c 69 71 75 6f 74 73 | 200 .l |aliquots|
|000059c0| 20 69 6e 20 31 2e 35 20 | 6d 6c 20 65 70 70 65 6e | in 1.5 |ml eppen|
|000059d0| 64 6f 72 66 73 20 62 79 | 20 66 72 6f 7a 65 6e 20 |dorfs by| frozen |
|000059e0| 69 6e 20 64 72 79 2d 69 | 63 65 20 66 69 72 73 74 |in dry-i|ce first|
|000059f0| 20 61 6e 64 20 74 68 65 | 6e 20 73 74 6f 72 65 64 | and the|n stored|
|00005a00| 20 69 6e 20 2d 20 38 30 | 20 a1 43 20 66 72 65 65 | in - 80| .C free|
|00005a10| 7a 65 72 2e 0d 0d 45 6c | 65 63 74 72 6f 70 6f 72 |zer...El|ectropor|
|00005a20| 61 74 69 6f 6e 20 6f 66 | 20 45 6c 65 63 74 72 6f |ation of| Electro|
|00005a30| 2d 43 6f 6d 70 65 74 65 | 6e 74 20 43 65 6c 6c 73 |-Compete|nt Cells|
|00005a40| 3a 0d 45 71 75 69 70 6d | 65 6e 74 73 3a 0d 20 20 |:.Equipm|ents:.  |
|00005a50| 20 20 20 42 69 6f 2d 52 | 61 64 20 47 65 6e 65 20 |   Bio-R|ad Gene |
|00005a60| 50 75 6c 73 65 72 0d 20 | 20 20 20 20 42 69 6f 2d |Pulser. |    Bio-|
|00005a70| 52 61 64 20 50 75 6c 73 | 65 72 20 43 75 76 65 74 |Rad Puls|er Cuvet|
|00005a80| 74 65 20 30 2e 32 20 63 | 6d 20 67 61 70 0d 0d 43 |te 0.2 c|m gap..C|
|00005a90| 6f 6e 64 69 74 69 6f 6e | 3a 0d 20 20 20 20 20 56 |ondition|:.     V|
|00005aa0| 6f 6c 74 61 67 65 3a 20 | 32 2e 35 20 56 20 69 2e |oltage: |2.5 V i.|
|00005ab0| 65 2e 20 31 32 2e 35 20 | 56 2f 63 6d 0d 20 20 20 |e. 12.5 |V/cm.   |
|00005ac0| 20 20 43 61 70 61 63 74 | 61 6e 63 65 3a 20 32 35 |  Capact|ance: 25|
|00005ad0| 20 b5 46 0d 20 20 20 20 | 20 44 75 72 61 74 69 6f | .F.    | Duratio|
|00005ae0| 6e 3a 20 61 75 74 6f 0d | 0d 20 20 20 20 20 4f 6e |n: auto.|.     On|
|00005af0| 65 20 68 75 6e 64 72 65 | 64 20 b5 6c 20 6f 66 20 |e hundre|d .l of |
|00005b00| 65 6c 65 63 74 72 6f 2d | 63 6f 6d 70 65 74 65 6e |electro-|competen|
|00005b10| 74 20 63 65 6c 6c 73 20 | 77 61 73 20 6d 69 78 65 |t cells |was mixe|
|00005b20| 64 20 77 69 74 68 20 31 | 30 30 20 6e 67 20 6f 66 |d with 1|00 ng of|
|00005b30| 20 44 4e 41 20 61 6e 64 | 20 74 68 65 6e 20 73 75 | DNA and| then su|
|00005b40| 62 6a 65 63 74 65 64 20 | 74 6f 20 47 65 6e 65 20 |bjected |to Gene |
|00005b50| 50 75 6c 73 65 72 2e 20 | 54 77 6f 20 68 75 6e 64 |Pulser. |Two hund|
|00005b60| 72 65 64 20 b5 6c 20 6f | 66 20 53 4f 43 20 77 61 |red .l o|f SOC wa|
|00005b70| 73 20 61 64 64 65 64 20 | 74 6f 20 74 68 65 20 63 |s added |to the c|
|00005b80| 75 76 65 74 74 65 20 74 | 6f 20 72 65 63 6f 76 65 |uvette t|o recove|
|00005b90| 72 20 74 72 61 6e 73 66 | 6f 72 6d 65 64 20 63 65 |r transf|ormed ce|
|00005ba0| 6c 6c 73 2e 20 54 68 65 | 20 65 6c 65 63 74 72 6f |lls. The| electro|
|00005bb0| 70 6f 72 61 74 65 64 20 | 63 65 6c 6c 73 20 77 65 |porated |cells we|
|00005bc0| 72 65 20 61 6c 6c 6f 77 | 65 64 20 74 6f 20 72 65 |re allow|ed to re|
|00005bd0| 63 6f 76 65 72 20 69 6e | 20 31 20 6d 6c 20 53 4f |cover in| 1 ml SO|
|00005be0| 43 20 66 6f 72 20 34 20 | 68 72 2e 20 28 32 20 67 |C for 4 |hr. (2 g|
|00005bf0| 65 6e 65 72 61 74 69 6f | 6e 20 74 69 6d 65 29 2e |eneratio|n time).|
|00005c00| 20 61 74 20 72 6f 6f 6d | 20 74 65 6d 70 2e 0d 20 | at room| temp.. |
|00005c10| 20 20 20 20 53 65 72 69 | 61 6c 20 64 69 6c 75 74 |    Seri|al dilut|
|00005c20| 69 6f 6e 20 6f 66 20 74 | 68 65 20 74 72 61 6e 73 |ion of t|he trans|
|00005c30| 66 6f 72 6d 61 6e 74 73 | 20 61 6e 64 20 61 70 70 |formants| and app|
|00005c40| 72 6f 70 72 69 61 74 65 | 20 63 6f 6e 74 72 6f 6c |ropriate| control|
|00005c50| 20 77 65 72 65 20 70 6c | 61 74 65 64 20 6f 75 74 | were pl|ated out|
|00005c60| 20 6f 6e 74 6f 20 73 65 | 6c 65 63 74 69 76 65 20 | onto se|lective |
|00005c70| 6d 65 64 69 75 6d 2e 0d | 2d 2d 2d 2d 2d 2d 2d 2d |medium..|--------|
|00005c80| 2d 2d 45 6c 65 63 74 72 | 6f 70 6f 72 61 74 69 6f |--Electr|oporatio|
|00005c90| 6e 20 6f 66 20 42 61 63 | 74 65 72 69 61 00 00 00 |n of Bac|teria...|
|00005ca0| 00 00 17 40 43 41 52 44 | 00 00 16 2a 00 00 00 00 |...@CARD|...*....|
|00005cb0| 00 00 00 00 00 00 00 00 | 00 00 00 00 00 00 00 00 |........|........|
|00005cc0| 00 00 0b b6 00 00 0a ab | 00 00 00 00 00 00 00 00 |........|........|
|00005cd0| 00 01 00 00 16 dc 00 06 | 16 d7 80 0a 00 00 00 02 |........|........|
|00005ce0| 00 1c 00 03 4e 75 74 72 | 69 74 69 6f 6e 61 6c 20 |....Nutr|itional |
|00005cf0| 4d 75 74 61 6e 74 20 49 | 73 6f 6c 61 74 69 6f 6e |Mutant I|solation|
|00005d00| 0d 0d 42 61 63 6b 67 72 | 6f 75 6e 64 3a 0d 0d 20 |..Backgr|ound:.. |
|00005d10| 20 46 72 6f 6d 20 6c 61 | 72 67 65 20 70 6f 70 75 | From la|rge popu|
|00005d20| 6c 61 74 69 6f 6e 73 20 | 6f 66 20 62 61 63 74 65 |lations |of bacte|
|00005d30| 72 69 61 2c 20 72 61 72 | 65 20 6d 75 74 61 6e 74 |ria, rar|e mutant|
|00005d40| 20 63 65 6c 6c 73 20 77 | 68 69 63 68 20 63 61 6e | cells w|hich can|
|00005d50| 20 73 75 72 76 69 76 65 | 20 61 6e 64 20 67 72 6f | survive| and gro|
|00005d60| 77 20 6f 6e 20 61 20 6d | 65 64 69 75 6d 20 77 68 |w on a m|edium wh|
|00005d70| 69 63 68 20 72 65 73 74 | 72 69 63 74 73 20 74 68 |ich rest|ricts th|
|00005d80| 65 20 67 72 6f 77 74 68 | 20 6f 66 20 74 68 65 20 |e growth| of the |
|00005d90| 27 77 69 6c 64 2d 74 79 | 70 65 27 20 70 61 72 65 |'wild-ty|pe' pare|
|00005da0| 6e 74 20 73 74 72 61 69 | 6e 20 63 61 6e 20 72 65 |nt strai|n can re|
|00005db0| 61 64 69 6c 79 20 62 65 | 20 69 73 6f 6c 61 74 65 |adily be| isolate|
|00005dc0| 64 2e 20 46 6f 72 20 65 | 78 61 6d 70 6c 65 2c 20 |d. For e|xample, |
|00005dd0| 63 6f 6c 6f 6e 69 65 73 | 20 64 65 72 69 76 65 64 |colonies| derived|
|00005de0| 20 66 72 6f 6d 20 63 65 | 6c 6c 73 20 77 68 69 63 | from ce|lls whic|
|00005df0| 68 20 68 61 76 65 20 6d | 75 74 61 74 65 64 20 74 |h have m|utated t|
|00005e00| 6f 20 64 72 75 67 20 72 | 65 73 69 73 74 61 6e 63 |o drug r|esistanc|
|00005e10| 65 20 63 61 6e 20 62 65 | 20 73 65 6c 65 63 74 65 |e can be| selecte|
|00005e20| 64 20 62 79 20 70 6c 61 | 74 69 6e 67 20 6c 61 72 |d by pla|ting lar|
|00005e30| 67 65 20 70 6f 70 75 6c | 61 74 69 6f 6e 73 20 6f |ge popul|ations o|
|00005e40| 66 20 73 65 6e 73 69 74 | 69 76 65 20 62 61 63 74 |f sensit|ive bact|
|00005e50| 65 72 69 61 20 6f 6e 20 | 6d 65 64 69 61 20 63 6f |eria on |media co|
|00005e60| 6e 74 61 69 6e 69 6e 67 | 20 74 68 65 20 64 72 75 |ntaining| the dru|
|00005e70| 67 2e 20 53 69 6d 69 6c | 61 72 6c 79 2c 20 6e 75 |g. Simil|arly, nu|
|00005e80| 74 72 69 74 69 6f 6e 61 | 6c 20 69 6e 64 65 70 65 |tritiona|l indepe|
|00005e90| 6e 64 65 6e 63 65 20 6f | 66 20 61 20 67 72 6f 77 |ndence o|f a grow|
|00005ea0| 74 68 20 66 61 63 74 6f | 72 20 72 65 71 75 69 72 |th facto|r requir|
|00005eb0| 65 64 20 62 79 20 74 68 | 65 20 70 61 72 65 6e 74 |ed by th|e parent|
|00005ec0| 61 6c 20 73 74 72 61 69 | 6e 20 63 61 6e 20 62 65 |al strai|n can be|
|00005ed0| 20 73 65 6c 65 63 74 65 | 64 20 62 79 20 70 6c 61 | selecte|d by pla|
|00005ee0| 74 69 6e 67 20 6c 61 72 | 67 65 20 70 6f 70 75 6c |ting lar|ge popul|
|00005ef0| 61 74 69 6f 6e 73 20 6f | 66 20 63 65 6c 6c 73 20 |ations o|f cells |
|00005f00| 6f 6e 20 6d 65 64 69 61 | 20 6c 61 63 6b 69 6e 67 |on media| lacking|
|00005f10| 20 74 68 65 20 67 72 6f | 77 74 68 20 66 61 63 74 | the gro|wth fact|
|00005f20| 6f 72 2e 0d 09 20 4e 75 | 74 72 69 74 69 6f 6e 61 |or... Nu|tritiona|
|00005f30| 6c 6c 79 2d 64 65 66 69 | 63 69 65 6e 74 20 28 61 |lly-defi|cient (a|
|00005f40| 75 78 6f 74 72 6f 70 68 | 69 63 29 20 6d 75 74 61 |uxotroph|ic) muta|
|00005f50| 6e 74 73 20 6f 62 76 69 | 6f 75 73 6c 79 20 63 61 |nts obvi|ously ca|
|00005f60| 6e 6e 6f 74 20 62 65 20 | 69 73 6f 6c 61 74 65 64 |nnot be |isolated|
|00005f70| 20 69 6e 20 74 68 69 73 | 20 77 61 79 2e 20 41 20 | in this| way. A |
|00005f80| 6d 65 74 68 6f 64 20 66 | 61 63 69 6c 69 74 61 74 |method f|acilitat|
|00005f90| 69 6e 67 20 74 68 65 20 | 69 73 6f 6c 61 74 69 6f |ing the |isolatio|
|00005fa0| 6e 20 6f 66 20 74 68 65 | 73 65 20 6d 75 74 61 6e |n of the|se mutan|
|00005fb0| 74 73 2c 20 64 65 76 65 | 6c 6f 70 65 64 20 62 79 |ts, deve|loped by|
|00005fc0| 20 44 61 76 69 73 20 28 | 31 39 34 38 29 2c 20 69 | Davis (|1948), i|
|00005fd0| 73 20 62 61 73 65 64 20 | 6f 6e 20 74 68 65 20 66 |s based |on the f|
|00005fe0| 61 63 74 20 74 68 61 74 | 20 70 65 6e 69 63 69 6c |act that| penicil|
|00005ff0| 6c 69 6e 20 6b 69 6c 6c | 73 20 6f 6e 6c 79 20 67 |lin kill|s only g|
|00006000| 72 6f 77 69 6e 67 20 62 | 61 63 74 65 72 69 61 2e |rowing b|acteria.|
|00006010| 20 43 65 6c 6c 73 20 6f | 66 20 74 68 65 20 6e 75 | Cells o|f the nu|
|00006020| 74 72 69 74 69 6f 6e 61 | 6c 6c 79 20 6e 6f 6e 2d |tritiona|lly non-|
|00006030| 72 65 71 75 69 72 69 6e | 67 20 28 70 72 6f 74 6f |requirin|g (proto|
|00006040| 74 72 6f 70 68 69 63 29 | 20 77 69 6c 64 2d 74 79 |trophic)| wild-ty|
|00006050| 70 65 20 73 74 72 61 69 | 6e 20 6f 66 20 45 2e 20 |pe strai|n of E. |
|00006060| 63 6f 6c 69 20 4b 31 32 | 20 77 69 6c 6c 20 67 72 |coli K12| will gr|
|00006070| 6f 77 20 69 6e 20 61 20 | 6d 65 64 69 75 6d 20 63 |ow in a |medium c|
|00006080| 6f 6e 74 61 69 6e 69 6e | 67 20 6f 6e 6c 79 20 69 |ontainin|g only i|
|00006090| 6e 6f 72 67 61 6e 69 63 | 20 73 61 6c 74 73 20 61 |norganic| salts a|
|000060a0| 6e 64 20 67 6c 75 63 6f | 73 65 20 28 6d 69 6e 69 |nd gluco|se (mini|
|000060b0| 6d 61 6c 20 6d 65 64 69 | 75 6d 29 2c 20 61 6e 64 |mal medi|um), and|
|000060c0| 20 61 72 65 20 6b 69 6c | 6c 65 64 20 69 66 20 70 | are kil|led if p|
|000060d0| 65 6e 69 63 69 6c 6c 69 | 6e 20 69 73 20 70 72 65 |enicilli|n is pre|
|000060e0| 73 65 6e 74 2e 20 4e 75 | 74 72 69 74 69 6f 6e 61 |sent. Nu|tritiona|
|000060f0| 6c 6c 79 2d 64 65 66 69 | 63 69 65 6e 74 20 6d 75 |lly-defi|cient mu|
|00006100| 74 61 6e 74 73 20 77 68 | 69 63 68 20 63 61 6e 6e |tants wh|ich cann|
|00006110| 6f 74 20 67 72 6f 77 20 | 69 6e 20 74 68 69 73 20 |ot grow |in this |
|00006120| 6d 65 64 69 75 6d 20 77 | 69 6c 6c 20 73 75 72 76 |medium w|ill surv|
|00006130| 69 76 65 20 74 68 69 73 | 20 70 65 6e 69 63 69 6c |ive this| penicil|
|00006140| 6c 69 6e 20 74 72 65 61 | 74 6d 65 6e 74 2e 20 54 |lin trea|tment. T|
|00006150| 68 65 20 61 64 64 69 74 | 69 6f 6e 20 6f 66 20 70 |he addit|ion of p|
|00006160| 65 6e 69 63 69 6c 6c 69 | 6e 20 74 6f 20 67 72 6f |enicilli|n to gro|
|00006170| 77 69 6e 67 20 63 75 6c | 74 75 72 65 73 20 6f 66 |wing cul|tures of|
|00006180| 20 63 65 6c 6c 73 20 74 | 68 65 72 65 66 6f 72 65 | cells t|herefore|
|00006190| 20 69 6e 63 72 65 61 73 | 65 73 20 74 68 65 20 70 | increas|es the p|
|000061a0| 72 6f 70 6f 72 74 69 6f | 6e 20 61 6d 6f 6e 67 20 |roportio|n among |
|000061b0| 74 68 65 20 73 75 72 76 | 69 76 6f 72 73 20 6f 66 |the surv|ivors of|
|000061c0| 20 61 6e 79 20 61 75 78 | 6f 74 72 6f 70 68 69 63 | any aux|otrophic|
|000061d0| 20 6d 75 74 61 6e 74 73 | 20 74 68 61 74 20 6d 61 | mutants| that ma|
|000061e0| 79 20 62 65 20 70 72 65 | 73 65 6e 74 2e 0d 09 20 |y be pre|sent... |
|000061f0| 41 75 78 6f 74 72 6f 70 | 68 69 63 20 6d 75 74 61 |Auxotrop|hic muta|
|00006200| 6e 74 73 20 69 6e 20 45 | 2e 20 63 6f 6c 69 20 4b |nts in E|. coli K|
|00006210| 31 32 20 61 72 65 20 75 | 73 75 61 6c 6c 79 20 70 |12 are u|sually p|
|00006220| 72 65 73 65 6e 74 20 69 | 6e 20 76 65 72 79 20 6c |resent i|n very l|
|00006230| 6f 77 20 6e 75 6d 62 65 | 72 73 20 28 61 62 6f 75 |ow numbe|rs (abou|
|00006240| 74 20 31 20 69 6e 20 31 | 30 37 29 20 62 75 74 20 |t 1 in 1|07) but |
|00006250| 74 68 69 73 20 70 72 6f | 70 6f 72 74 69 6f 6e 20 |this pro|portion |
|00006260| 63 61 6e 20 62 65 20 63 | 6f 6e 73 69 64 65 72 61 |can be c|onsidera|
|00006270| 62 6c 79 20 69 6e 63 72 | 65 61 73 65 64 20 62 79 |bly incr|eased by|
|00006280| 20 74 72 65 61 74 69 6e | 67 20 61 20 63 75 6c 74 | treatin|g a cult|
|00006290| 75 72 65 20 77 69 74 68 | 20 61 20 6d 75 74 61 67 |ure with| a mutag|
|000062a0| 65 6e 20 28 6d 75 74 61 | 67 65 6e 65 73 69 73 29 |en (muta|genesis)|
|000062b0| 2e 0d 09 20 54 68 65 20 | 6d 75 74 61 67 65 6e 20 |... The |mutagen |
|000062c0| 6f 66 20 63 68 6f 69 63 | 65 20 69 73 20 45 74 68 |of choic|e is Eth|
|000062d0| 79 6c 20 4d 65 74 68 61 | 6e 65 73 75 6c 66 6f 6e |yl Metha|nesulfon|
|000062e0| 61 74 65 20 28 45 4d 53 | 29 2e 20 55 6e 64 65 72 |ate (EMS|). Under|
|000062f0| 20 63 65 72 74 61 69 6e | 20 63 6f 6e 64 69 74 69 | certain| conditi|
|00006300| 6f 6e 73 20 61 20 67 72 | 65 61 74 20 70 72 6f 70 |ons a gr|eat prop|
|00006310| 6f 72 74 69 6f 6e 20 28 | 61 62 6f 75 74 20 32 30 |ortion (|about 20|
|00006320| 25 29 20 6f 66 20 74 68 | 65 20 73 75 72 76 69 76 |%) of th|e surviv|
|00006330| 6f 72 73 20 6f 66 20 45 | 4d 53 20 74 72 65 61 74 |ors of E|MS treat|
|00006340| 6d 65 6e 74 20 6d 61 79 | 20 62 65 20 61 75 78 6f |ment may| be auxo|
|00006350| 74 72 6f 70 68 69 63 2e | 20 41 66 74 65 72 20 6d |trophic.| After m|
|00006360| 75 74 61 67 65 6e 65 73 | 69 73 2c 20 74 68 65 20 |utagenes|is, the |
|00006370| 74 72 65 61 74 65 64 20 | 63 65 6c 6c 73 20 61 72 |treated |cells ar|
|00006380| 65 20 73 75 62 2d 63 75 | 6c 74 75 72 65 64 20 69 |e sub-cu|ltured i|
|00006390| 6e 20 62 72 6f 74 68 20 | 74 6f 20 61 6c 6c 6f 77 |n broth |to allow|
|000063a0| 20 74 68 65 20 73 65 67 | 72 65 67 61 74 69 6f 6e | the seg|regation|
|000063b0| 20 6f 66 20 6d 75 74 61 | 6e 74 20 61 6e 64 20 6e | of muta|nt and n|
|000063c0| 6f 6e 2d 6d 75 74 61 6e | 74 20 6e 75 63 6c 65 69 |on-mutan|t nuclei|
|000063d0| 20 66 72 6f 6d 20 6d 75 | 6c 74 69 2d 6e 75 63 6c | from mu|lti-nucl|
|000063e0| 65 61 74 65 20 63 65 6c | 6c 73 20 61 6e 64 20 74 |eate cel|ls and t|
|000063f0| 6f 20 70 65 72 6d 69 74 | 20 74 68 65 20 70 68 65 |o permit| the phe|
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