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I3
ENTER ONE OF THE FOLLW ING: TO BEGIN FOLDING: TO QUIT THE PRDGWYl TO SET W Y OF THE DEFAULT P A M E T E R S
FOLDING BASES 1 TO -16.9 ENERGY =
7 2 OF
ie 28 38 CGGWTATGCTCG AGT TAC ACA CTWTACWTTGC T TGT WCTTTATGGTTMCG G WGTWCCA --G TAC 78 68 58 48 ENTER: T T E R H I M T E , NS NEW SEQUENCE, NF NEW FRAGMENT, 0 OLTPUT PARAMETER 1 Fy.(O 72. D E F IN IT ION , OR THE ENDPOINTS OF A SUBFWIMENT BETWEEN E D WITH 1 TO FORCE ENDS TO BASEPAIR.
_---
4
FORTRPN STOP
S h a l l we d o t h i s a g a i n ? ( Y / N ) : h
You a r e n w l e a v i n g RNAFOLD.
FIG. 10. Output from RNAFOLD, a nucleic acid folding program. The original program was generously provided by its author, Michael Zuker, Canadian Research Council, and has been integrated into DNACE.
182
JOSEPH L. MODELEVSKY
You h a v e j u s t e n t e r e d HYDROPATH. D o y o u w a n t a d e s c r i p t i o n of
t h e program?
You h a v e j u s t e n t e r e d HYDROPATH. HYDROPATH p r o g r e s s i v e l y e v a l u a t e s t h e h y d r o p h i l i c i t Y a n d h y d r o p h o b i c i t y o f a p r o t e i n a l o n g i t s amino a c i d sequence. HYDROPATH UECE a m o v i n g s e g n e n t a p p r o a c h t o c o n t i n u o u s l y d e t e r m i n e t h e a u e r a g e HYDROPATHy w i t h i n a s e g n e n t a s i t a d v a n c e s t h r o u g h t h e segm ent a n d c o m p a r e s t h e s e a v e r a g e d s e g n e n t t o t h e w h o l e p r o t e i n HYDROPATHy. I n t e r i o r segments o f t h e p r o t e i n s h o u l d E x t e r i o r segnrntc c o r r e s p o n d t o t h e h y d r p h o b i c s i d e of t h e o u t p u t r a n g e . s h o u l d c o r r e s p o n d t o t h e h y d r o p h y l l i c s i d e of t h e o u t p u t r a n g e . ( J . KYTE a n d R . DOOLITTLE, J.Mol. E i o l . 157, 185-132, 1982). You MUST USE ONE-LETTER a m i n o a c i d c o d e s . Code c o n v e r s i o n i s p e r m i t t e d i n t h e p r o g r a m . The r i g h t s i d e of t h r p l o t i s HYDROPHOBIC. The l e f t s i d e o f t h e p l o t i s HYDROPHILIC. F o r an m i n o a c i d f i l e E n t e r t h e f i l e name: )-it
(i.e
SAnPLE.W),
1s t h e a a f i l e i n 1 o r 3 l e t t e r c o d e s 7 ( e n t e r E n t e r t w o c(A id’s:
1 o r 3):m
i n p u t l e n g t h of hydropath se e n t over which to average, (n u mbe r o f aa r e s i d u e s ) : Want a P l o t ? Want a p r e d i c t i o n of s e c o n d a r y s t r u c t u r e ? Llr) You h a w J U S ~e n t e r e d a v e r s i o n o f t h e Fasman A n a l y s i s . T h i s u e r r i o n p r e d i c t s s e c o n d a r y s t r u c t u r e c h a r a c t e r i s t i c s of t h e a m i n o a c i d sequence i n p u t i n HYDROPATH. The c a l c u l a t i o n s a r e b a s e d u p o n c o n f o r m a t i o n a l p a r a m e t e r s e s t a b l i s h e d f o r 29 r e f e r e n c e p r o t e i n s : Reference Set I
-
Chou a n d Fasman, B i o c h e m 13, p.211- 245 1974 a n d A d v a n c e s i n E n z y m o l o g y , 1978; R e f e r e n c e S e t 2 = FEBS L e t t e r s 93, 19-24, 1978.
FIG. 11. Output from program HYDROPATH. The output contains a hydropathicity analysis of the amino terminus of the OmpF protein of Escherichia coli
exist, but there may be limited data on the protein itself. The target gene product may be associated with a cloned segment of DNA, but the resolution of the DNA into hnctional domains is not a simple problem to resolve without more data. The computer can assist in the analysis of newly cloned DNA. Once a clone has been isolated, the cloned DNA sequence is readily determinable. Some useful computational tools which can be applied to analyze such DNA sequences include searches for open reading frames, determinations of coding probability for those open reading frames (Fickett, 1982), and searches for functional elements (promoters, ribosome-binding sites, terminators).
183
COMPUTER APPLICATIONS IN GENETIC ENGINEERING
12)
E n t e r r e f e r e n c e s e t number ( I o r 2 ) : T r a n s l a t i n g I l e t t e r codes t o 3 l e t t e r s . 368 codes. E n t e r P r o t e i n Name i n c a p i t a l
l e t t e r s (e.g.
l i m i t 20 l e t t e r s : [ E . c o l i mpF]
W E ) ,
T r a n s l a t i n g 3 l e t t e r codes t o I l e t t e r . 368 codes ( 1888 c h a r a c t e r s ) .
I 2 3 4
5 6
7 8 9 18
II 12 13 14 15 16
17 18 19 28 21 22 23 24
25 26
27
8.8 8.8 8.8 31.5 33.8 42.2 58.9 52.8 58.1 58.1 52.8 52.8 49.3 44.7 45.6 45.6 38.4 36.5 34.1 28.8 33.7 33.1
ti o K o R o N
" t
0
I
a *
0
L A V
a ^
0 0
I
0
v
0 o 0 0
-
a -
o
P A L L V A G T A N A A
1
a a ^
3 -
0 0 0 0 0 0 0 0
27.8 E 27.4 I 22.1 Y 19.9 N 19.9 K
a -
o
a
a a ^
+
0. 0. 0 0 0
i
t t
FIG. 11. (Continued)
These analytical results can suggest the necessary experiments to confirm assignments of functional domains. 4. Display of Genetic Z n f o m t i o n All of the analyses described above create output which must be interpreted by the researcher. The information generated by these analyses must somehow be displayed for review. Computerized display of genetic information has been addressed in a number of ways. Simple data tables containing a list of information (e.g., strain designations and their genetic markers) may be used. Restriction maps may be displayed linearly across a screen, with cut sites delineated by labeled or unlabeled tick marks, or as circular maps. For the genetic engineer, the circular restriction map is the
184
JOSEPH L. MODELEVSKY
predominant format for display of plasmid genetic information. Circular restriction maps frequently contain a large amount of information, including dozens of restriction sites, genetic markers, and statistical and historical information; however, such maps do not readily lend themselves to computerized display. Often, sites of interest overlap or are congested into small display areas; circles themselves require relatively high graphic resolution for display. Recently, storage, retrieval, and display of circular restriction maps have been implemented in DNACE using the program PLASMAP (Stone et al., 1984). PLASMAP forms the basis for the graphic display of color-coded genetic information which is superimposed upon conventional line displays. Using this approach, a variety of analytical results and constraints can be displayed on a single screen of information (Fig. 12).
PSEUDO p B R 3 2 2 D e r i v a t i v e Clal Hincl I Rrul
Pvul
, Xmal I I Nrul
1000
Sonple PLASYAP doto flle ond dlsploy. A alnple olpho-nummrlc toble Is convmrtmd lnto o grophfc dlrploy of o convmntlonol. tnformotlon-dmn#e, cfrculor rertrlctlon m a p . Thfs mop lncluder locatlonr o f reverol known restrtctlon enrym recognltlon sequmncer occurlng two or fewer timer ln pBR322DNA. The poaltlona of the tetrocycllnm ond omploillfn rmrtrtoncm goner: romplms of overlopplng Iondmorkr and o pmeudo cloned erprmsrlon unlt (Px. pronotrr X; RBS. rlboronm blndlng slte: Gene X ) orm olro Indlcatmd. Plotted on HewImttPcokord 7220 plotter. Rmstrlctlon rltes from Sutcllffe. J.G.. Cold Spring Horbor Symposlum (1979) 43. 77-90.
FIG. 12. Sample display from PLASMAP, the storage, edit, and retrieval system for circular restriction maps. Color-coded information may be superimposed on graphic display.
COMPUTER APPLICATIONS I N GENETIC ENGINEERING
185
C . A SAMPLEAPPLICATION Thus far, I have dealt mainly with the computational side of computer applications in genetic engineering. Next, to describe how genetic engineers might operate, I wish to specifically demonstrate the applications described above via a model genetic engineering project. The target gene in this model is the hypothetical human INSIGHT gene. Assume that the INSIGHT gene product is expressed in the pituitary at barely detectable levels and is believed to have a molecular weight of approximately 9000. The demonstration begins with the only known sequence, 21 amino acids at the carboxyl terminus of the protein (Fig. 13). The decision has been made to clone INSIGHT cDNA. To begin, RNA is extracted from human pituitaries, and from this RNA preparation, a fraction of poly(A) (messenger) RNA is isolated. This poly(A) RNA will be the template for cDNA synthesis. To increase the specificity of the synthesized cDNA population, a cDNA synthesis primer specific for INSIGHT messenger RNA will be used in lieu of an oligo(dT) primer. To design this DNA primer, the computer will reverse-translate the known INSIGHT amino acid sequence and select the coding region of minimal degeneracy (i.e., the region coded for by a minimum number of possible DNA sequences). Then a set of synthetic oligonucleotides (DNA pieces) complementary to this coding region will be made; these oligonucletides will be used as primers for cDNA synthesis. Since the known amino acid sequence is at the carboxyl terminus of INSIGHT protein (i.e., coded from the 3’ end of the message), using a primer for this region should provide highly discrete, nearly full-length, cDNAs for INSIGHT protein. As shown‘ in Fig. 14, there is a region of the INSIGHT amino acid sequence (beginning at amino acid number 9 of the known 21) that may be coded for by 8 different 12-mers (12 base long oligonucleotides) or 16 different 14-mers. A 12-mer should be capable of priming cDNA synthesis, should be highly discriminatory in the RNA population, and may be used as a colony hybridization probe to screen resultant clones for the presence of INSIGHT cDNA. (Colony hybridization is a technique which may identify bacterial colonies which contain “probed-for’’ DNA sequences.) To use these oligonucleotides as probes in a colony hybridization screen-
Carboxy t e r m i n u s of hypothetical I N S I G H T protein.
-_---h i n o
a c i d sequence
S t a r t i n g point
1.
I L E T R P MET GLY -- A S P ARG PHE PRO ARG _-_-__-_- -__ THR -_____ __---- --- -- --- -__-____-__GLY I L E MET ARG PRO -- THR __---_ --_ -_- -_- _- --_
I V A L GLY ALA THR SER
_ _ -___
16
Frc. 13. The carboxyl terminus of the hypothetical INSIGHT protein.
186
JOSEPH L. MODELEVSKY i**I+.*l****C*I******f+************************~*********ff*
APROF You h a v e j u s t e n t e r e d APROF: APROF s c a n s s u b s e q u e n c e s i n an M s t r i n g a n d g e n e r a t e s a s e t o f DNA s e g m e n t s w h i c h c o u l d code f o r t h o s e subsequenes. The D M s e q u e n c e s w i t h t h e h i g h e s t number o f f i x e d b a s e s o r GC c o n t e n t a n d minimum number of DNA c h a i n s r e q u i r e d t o c o d e f o r t h a t M subsequence i s d e t e r m i n e d a n d p r i n t e d . Useful i n the design o f s p e c i f i c DNCI p r o b e s +or unknown gene s e q u e n c e s b a s e d u p o n known M sequences.
*ii*t~****+**si*ra***********m***4************~******~***********
Comnands:
READ t o r e a d an aa f i l e SCAN t o compute f i x e d b a s e s a n d p o s s i b l e sequences DNA t o d i s p l a y t h e c o d o n s f o r a segment Ll ST d i s p l a y s t h e a m i n o a c i d sequence ENTER i n p u t an a m i n o a c i d sequence f r m the teletype. PRINT e n a b l e s o u t p u t t o t h e l i n e p r i n t e r NOPF.INT d i s a b l e s l i n e p r i n t e r o u t p u t
+a
F o r an a m i n o a c i d f i l e E n t e r t h e f i l e name: i n s i h t f . a a a E n t e r t w o PA id‘s: Translate 1 l e t t e r t o l e t t e r codes’ T r a n s l a t e 3 l e t t e r t o I l e t t e r codes’
&-
The p e p t i d e i s The pseudo-dna
QOCJ
R
21 a m i n o a c i d s l o n g . f r o m bases 1 to
63.
F u l l listing’ O p t i m i z e C h a i n s , F i x e d Bases, o r GC c o n t e n t 7 C C F G I : m L e n g t h ( i n c o d o n s ) o f amina a c i d s e g n e n t t o be c o n s i d e r e d : E n t e r l o w c o d o n , h i g h codon:
I 2 6 7 The The The The
Fixed = Fixrd = Fixrd = Fixed =
segnent maximum maximum minimum
6 6 7 8
GC GC GC GC
=
=
6.42993 8.37837
E n t e r t h e l o w amino a c i d l o c a t i o n , Fixed =
19
GC
Chains = Chains = Chains = Chains =
64 64 12 3
3 8 6.72222 3
length is: number o f f i x e d b a s e s i s : gc c o n t e n t i s : number o f c h a i n s i s :
m
5
= 8.72222 = 8.72222
a
length of s e g m e n t : m
= 8.51667
Chains
=
27648
4 THR SER THR I L E TRP MET GLY ASP ARG PHE PRO ARC
ACC AGT ACC ATC ACG TCA ACG ATT ACT TCC ACT TCG TCT
II L
I
CCG CGL, CCT CGC CGG CGT
Fic;. 14. Computer-assisted INSIGHT primerlprobe design. Minimally redundant coding region is enclosed in box.
187
COMPUTER APPLICATIONS IN GENETIC ENGINEERING
D o you want t o do a h m o l o g y ( h y b r i d i z a t i o n ) t e s t o r a s i m p l e s t r i n g match’ Answer H o r H : F o r t h e s t r i n g YOU want t o s e a r c h , t h e base CNA f i l e E n t e r t h e f i l e name. p b r 3 2 2 dna E n t e r two DNCI i d , s : ’ F ’ 1 to The DNA c o n t a i n s 4363 b a s e s ( l o c a t i o n F o r t h e s t r i n g YOU want t o s e a r c h w i t h , t h e M S K f i l e , E n t e r t h e f i l e name:lprobes.dna\ E n t e r two 1.5’5: hasps 1 to The M S K c o n t a i n s E n t e r minimum l e n g t h and p e r c e n t : Output t o the l i n e p r i n t e r ’ 4363 The W s t r i n g goes from l o c a t i o n 1 to E n t e r low and h i g h l o c a t i o n s f o r DNA s e g 1 : 1 to 28 The M S K s t r i n g goes f r o m l o c a t i o n E n t e r l o w a n d h i g h l o c a t i o n s f o r M S K s e g 2: L i s t the input s t r i n g s ?
-
m28 :m
4363)
28)
:m
Beginning search.
I n d i c e s are
From 443 From 1 ATCGCCWWTC PTCNCCCATC
to to
454. 18.
From 888 From 1 GTCCCGCCACCA PTCNCCCATCCA
to to
899.
Fran 3523 3 From CGCCTCCATCCA CNCCCATCCA
1
4363
28
1
Len
12.
12.
P c t 9.83333 Len
12.
to to
3534. 12.
P c t 8.83333 Len
12.
to to
542. 28.
P c t 9.83333 Len
12.
P c t 8.83333
I@: From 531 Fran 17 TGGCCGGGGWC TGGATGGGNGAQ
3875 t o 3887. From From 17 t o 28. P c t 8 . 8 4 6 1 5 Len TGTATGCGGCGAC TGWTGGWGAQ 28: 5 homologies found. There were DNA c o n t a i n s 4363 b a s e s ( l o c a t i o n The The MASK c o n t a i n s 28 b a s e s ( l o c a t i o n
13.
1 to I to
4363)
28)
FIG. 15. Homology test between cloning vector pBR322 and INSIGHT prirner/probes.
ing process, there can be no homology between the probes and pBR322, the selected cloning vector. The results of a homology analysis reveal that there is no significant homology between the primer-probes and either strand of the vector (Fig. 15).Therefore, the eight 12-mer primers will be synthesized for use in the project. The oligonucleotide database is checked for the availability of precursors; none is found, so an automated synthesis machine is set up according to the formulations programmed into the synthesis management program library by the chemists (Fig. 16). Next, the cDNA is chemically synthesized. The progress of the synthesis
188
JOSEPH L. MODELEVSKY
T H I S PROGRAM W I L L CALCULATE THE AMOUNTS OF NLICLEOTIDE MONOMERS AND CONDENSING REAGENT TO DO A SAM RUN, THE SEQUENCE W I L L BE STORED I N ‘WWILOG.LIS BY RUNNING 0DD.EXE.
AND CAN BE ACCESSED
ENTER THE SEQUENCE TO BE SYNTHESIZED I N THE 5‘
TO 3‘
I F THERE ARE ANY M I X E D S I T E S , USE ‘P‘ ’N’ FOR A/C/G/T.
‘12‘ FOR A/G,
TYPE I N THE I D E N T I F Y I N G NCIME, USE, W D SYNTHESIS METHOD
NNWNNNNM‘I
e.g.
SEQUENCE’
PROBE
GGH-I
FOR T/C,
REQUESTOR,
SMITP
DIRECTION. AND
NOTEBOOK REFERENCE,
im-e-em
SAM
IGT CTCCCATCCA?
OTHER lNFDRMATION7 DO YOU WANT T H I S SEQUENCE WRITTEN I N ‘DNALOG’? W I L L SFIM BE DOING T H I S SYNTHESIS7 Y ’ N ’ D CCTACCCTCTG T H I S SEQUENCE IS A
12
mer.
A= C= F= T=
mL mL mL mL
OF OF OF OF
248
846 248 488
mg I N mg I N my IN
mg I N
2.4 8.4 2.4 4.8
Y / N 7 m
PYRIDINE. PYRIDINE. PYRIDINE. PYRIDINE.
THE AMOWT OF MSNT NEEDED IS:
2.5
g OF MSNT I N 15.2
mL OF A C E T O N I T R I L E .
.....................................................................
d3
DO YOU HAVE ANOTHER ENTRY’
ENTER Y ( e s )
or N c o ,
ENTER THE NEXT SEQUENCE. TYPE I N THE I D E N T I F Y I N G NAME, AND SYNTHESIS METHOD e.g.
NW“W
GGH-1
USE,
PROBE
REQCIESTOR, NOTEBOOK REFERENCE,
SMITH
CKR-8-88B
SAM
SEQUENCE’ IQTCNCCCATCCA) OTHER INFORMATIONT
DO YOLl WANT T H I S SEQUENCE WRITTEN I N ‘DNALOG W I L L SAM BE DOING T H I S SYNTHESIST Y/N7 Y CCTACCCNCTQ T H I S SEQUENCE IS A
0
Y/N’
N
12 m e r .
A= 248 m Q IN 2.4 m L OF P Y R I D I N E . C= 848 m g I N 8.4 m L OF P Y R I D I N E . T= 368 mg I N 3.6 mL OF P Y R I D I N E . 128 m g EACH OF A AND G I N 2.4 mL OF P Y R I D I N E . A/G= A/C/G/T= 68 m g EACH OF A, G, C, &ND T I N 2.4 mL OF P Y R I D I N E . THE AMOUNT OF MSNT NEEDED I S :
2.5
Q
OF MyJT
I N 15.2
mL OF A C E T O N I T R I L E .
..................................................................... DCI YOU HAVE ANOTHER ENTRY?
ENTER Y ( e s )
o r N(o>
’N
Fic 16. Computer-assisted recipe formulation for an automated DNA synthesis machine (Figure courtesy of C Brush )
COMPUTER APPLICATIONS IN GENETIC ENGINEERING
189
T H I S PROGRAM WILL EITHER CALCULATE THE NUMBER OF NUCLEOTIOES T A I L E D ONTO A TENPLATE DI.yI POPULATION OR WILL PREDICT THE COLNTS TO BE INCORPORATED FOR A GIVEN T A I L LENGTH WDER GIVEN EXPERIMENTAL C a J D I T l ONS
.
THE NWBER
-
R
-
I S THE NUMBER OF RESIDUES ADDED PER DNA END.
__-__--_--_--_---_ ---
--_
T W T I S , R = TFIIL LENGTH. NOTICE: You MUST a n w e r a l l q u e s t i o n s ! You may answer q u e s t i o n s w i t h the FIRST LETTER o f
the answer.
W E FUN, YOU TAILER, YOU! W i l l we c a l c u l a t e T A I L LENGTH ( R ) , ESTIMATE C O M S I n a g i v e n t a i l (E) or QUIT
(Q)??B
ALL T = N = M = D = H =
NEW WRIABLES? YES OR N O ? Q TC4 PPT'ABLE DPM PER T M W P L E NUCLEOTIDE CONCENTRATION CuMolar) I*I OF DNA TEMPLATE ( d a l ) C#l CONCENT.WTION I N RX M I X ( u g / u l ) LABEL PRESENT I N RX M I X ( u C i / u l )
P l e a s e e n t e r the necessary decay c o r r e c t i o n or c o u n t i n g e f f i c i e n c y c o r r e c t i o n (decimal f r a c t i o n l e s s than 1 . e ; e n t e r I f o r no correction?!TJ E n t e r VOLUME o f ASS4Y M P L E ( u l ) ?
ENTER T (DFWTCA S W P L E ) ? ~ R = 18.1393 NOT F, PAD JOB OF T A I L I N G , i f I may say so. W i l l we c a l c u l a t e T A I L LENGTH ( R ) , ESTIMATE COUNTS i n a g i v e n t a i l (E) or QUIT t Q ) ? ? D I HOPE YOU APPRECIATE THE CWPLEXITY OF THE CFILCULFITIONS I JUST MADE. I BET YOU COULDN'T MOVE YOUR ' T A I L ' T W T FAST!
FIG. 17. Output from a program which calculates the extent of reaction of terminal transferase and substrate.
is calculated using several of the programs found in the computation program library (see Fig. 7, above). Using the enzyme terminal transferase, the ends of the cDNA molecules are adapted for cloning into the PstI restriction enzyme site of our vector, pBR322; the extent of the adapting reaction is determined using one of the programs (Fig. 17). Bacteria are transformed with the adapted cDNA; 5000 colonies arise and are screened further. Colony hybridization, using a mixture of radioactively labeled probes synthesized above, discriminates 30 colonies out of the 5000 as positive for the presence of DNA homologous to one or more of the probes. The size of the cloned DNA is examined for each colony; the cloned DNA is smaller than expected, but acceptable for an initial cloning at approximately 100-200 base pairs each. Other experiments indicate that in the majority of these 30 clones, the cloned DNA is homologous to the probes. The DNA sequences are rapidly determined, and they reveal that the probe-homologous sequence TGGATGGGAGAC is contained at one end of several of the cloned DNAs (Fig. 18). A complete cDNA obviously was not
190
JOSEPH L. MODELEVSKY
EDPYSIN is the main DNA editor, capable of simulating all
Known DM manipulations. Type HELP for a list of available c m a n d s . Remember, you must READ in a C+S sequtnce to begin and must WRITE to a permanent filr to save changes. DO NOT WRITE OVER PRE-EXISTING F I L E W E S ’ Maximum D W string length 188888. Maximum substring length 1888. Type HELP for information.
-
D M > U Enter the file name: r Z t Enter two DM id’s: Defaulting to id’% = 1 , 1
Z
2
DM>m
Enter Icw and high location5 (e.9. 1,1598): Enter l o w and high locations (e.g. 1,1588): String id Length
1 to
1, 91
91
I
Starting point
4 5 t . I . 2 . 3 58 GTCTACGGCC ATACCACCCT WCGCGCCC GATCTCGTCT GATCTCGGM
.
51 to
91
1,
.
6
7
.
8
.
9
91 GCTMGCAGG GTCGGGCCTG GTTAGTACTT GGATGGOAGA C
FIG. 18. Listing of “experimentally determined INSIGHT cDNA sequence.
h i n o acids Total sequence length is 91 < 1 To 91) Display from and to locations, CLP,FII: Translate in a eingle frame or all 3 frames7 [ I or 31 Initial translation l o c , M number, TER/ALL/PART:l*!$ Displaying D W 1 1 91 m 1 1 91 28
19
GTC TAC GGC CAT ACC ACC CTG
48
38
mc
GCG
ccc
GAT CTC GTC TGA TCT CGG
UAL TYR GLY HIS THR THR LEU A S N ALA PRO ASP LEU WL ?3? SER ARG SER THR ALA I L E PRO PRO 73? THR ARG PRO ILE SER SER ASP LEU GLY LEU ARG PRO TYR H I S PRO GLU ARG ALA ARG SER ARG LEU I L E SER GLU S 18 1s 58
68
78
88
913
M G CTCI CIBC AGO GTC GGG CCT GGT T4G TAC TTG W T GGG AGF, C LYS LEU SER ARG W L PRO GLY ?2? TYR LEU ASP GLY CIRG ??? SER ?l? A M G L Y T E R GLY LEU W L S B TAR TRP MET GLY ASP ALA LYS G I N GLY ARG TRP LEU W L LEU GLY TRP FLU ??? 25 38 28
aa conversion canplet.!!
FIG. 19. Translation of “experimentally determined” INSIGHT cDNA sequence. Prediction of potential amino-terminal amino acid sequence. Residues matching previously determined INSIGHT amino acid sequence are underlined.
COMPUTER APPLICATIONS IN GENETIC ENGINEERING
191
synthesized, and some of the rapid sequencing may be in error, but the data can be analyzed further. The cloned and identified cDNA may be used to reprobe the human pituitary cDNA clones constructed above for a fulllength INSIGHT clone. In the meantime, the determined DNA sequence is electronically translated to reveal some of the previously unknown INSIGHT protein sequence upstream from the known 21 amino acids of the carboxyl terminus (Fig. 19). There are a few discrepancies between the starting amino acid sequence and what was translated via computer; there may be some frameshift errors in our DNA sequencing or some errors in the amino acid sequencing from the carboxyl terminus. Further investigation is called for, but, at least some potentially useful data have been generated. The question of whether the cloned segment of the INSIGHT gene is related to any known DNA sequence may be asked at this point. The experimentally determined INSIGHT DNA sequence is used in a computer program which will search the entire sequence database for sequences homologous to INSIGHT DNA (Fig. 20). The search reveals that a piece of DNA unrelated to INSIGHT (i.e., cDNA synthesized from 5 S ribosomal RNA), which happens to contain DNA homologous to one of the probes, has been cloned. Unfortunately, the probe design was defective and the project must begin again. Throughout this model project, the cloned sequence could have been examined repeatedly by eye, but still may have been unrecognizable as what it indeed was. However, in a matter of hours, the computer was able to identify the cloned DNA. In actual practice this analysis should be applied during initial probe design. I reserved this result until now to emphasize the enormous contribution that the electronic sequence databases and searching programs can make to applied genetic engineering efforts. Such programs, available only during the last few years, can trap cloning errors earlier and more easily than many other techniques. D. EXPERTSYSTEMS The computational tools examined thus far provide technical and design assistance via step-by-step application. The scientist determines what questions to ask, when to ask the questions, what to do with the result, and what actions to carry out on the basis of accumulated knowledge. The tools follow a programmed order of logical steps to answer the questions and provide the results. In essence, the scientist does the thinking and makes the decisions; the computer simply computes. Expert systems are quite different from the computational tools described above. An expert system is a computer system which attempts to emulate human thought. Such a system can logically reason and draw conclusions based
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JOSEPH L. MODELEVSKY
----C O N F I G U R E
DATfiBfiSE----
DRBB:CONP.G€NlOCT4.DAT ! H u r m a l i a n sequence e n t r i e s . DRBB:[W.GENIOCTS.DAT ! Other v e r t e b r a t e sequence e n t r i e s . DRB8:IW.GENlOCT6.WT ! I n v e r t e b r a t e sequence e n t r i e s . 4 DRBB:tW.GENIOCT7.WT ! P l a n t e n t r i e s ( i n c l u d i n g funpi and a l g a e ) . 5 0RBB:CDI’YI.GENIOCTE.WT ! Sequence e n t r i e s f o r e u l c a r y o t i c o r g a n e l l e s . 6 DRBB:[W.GENlOCT9.WT ! B a c t e r i a l sequence e n t r i e s . 7 DRBBICW.GENIOCT~~.DCIT ! S t r u c t u r a l l?kM sequence e n t r i e s . S DRBBaCW.GENIOCTl1.DPIT ! V i r a l swquence e n t r i e s . 9 DRB0:[W.GENIOCTl2.DPT ! Phape s e q u e n c e e n t r i e s . 18 DRBB:IW.OENIOCTI3.DAT I S y n t h e t i c a n d c h i m e r i c sequence e n t r i e s . D e l e t e any of these f i l e s ? For t h e s t r i n g y o u w a n t t o s e a r c h w i t h , t h e Mask f i l e , E n t e r t h e f i l e name: l i n s l g h t c . d n a ) E n t e r t w o DNA id‘s: D e f a u l t i n g t o id’s = I , 1 The Mask s t r i n g g o e s f r o m l o c a t i o n -2 t o 93. E n t e r l o w and h i g h l o c a t i o n s ( e . 9 . 1,1588): E n t e r l a w a n d h i p h l o c a t i o n s (e.9. 1,1580): -2, 93 E n t e r minimum l e n g t h a n d p e r c e n t : 98.90 BY h a u many bases s h o u l d t h e M.y, i n d i c e s b e i n c r e m e n t e d ? a Output to t h e l i n e p r i n t e r ? 1
2
3
a
----SAMPLE
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POSITIVES WMHPRT HAMMET I CH WIMMET 1 I CH MRRGJS DNA f r o m DNA f r o m There were
400 BP MJA 134 t o 229, Mask f r o m I40 t o 229, Mask f r o m 2 homologies f o u n d .
UPDATED 1 to 4 to
~w/ei/s3 Pct 93, P c t
93,
96.9, l e n lBB.0, len
96. 9B.
HUMAlATl HCm41AT2 HUMA 1A T 3
HUIAIAT4 HUMPLJn HUMPDHCI HUMPCJMC2 HUMPOMCB HUMPOMCBM
ECOl BSRNA2
ECOlBSRNAS ECORRldSB FSHRRSSSTR
FIG. 20. Whole database search to identify “experimentally determined” INSIGHT cDNA sequence. Excerpts of interactions and output are presented. The database is configured, the homology analysis applied, and results reported. Another program provides supporting information for the identified loci. Results reveal that cloned cDNA is actually not INSIGHT cDNA, but human 5 S ribosomal cDNA.
upon an input expert knowledge base. The logical reasoning process applies the problem-solving techniques of experts in the field of application. Expert systems have been successfully developed for a number of scientific disciplines. Buchanan (1982) has compiled an excellent bibliography of works in this area. The Stanford University MOLGEN project has developed some examples of expert systems for genetic engineering. A prototype automated experiment planner has been developed which uses a skeletal plan refinement
193
COMPUTER APPLICATIONS I N G E N E T I C ENGINEERING
FSHRRSS
120 BP RRNA 1 to 9 5 , MasK f r o m 1 to 9 5 , MasK f r o m I to 9 5 , MasK f r o m 3 homologies found.
UPDATED -2 t o -1 t o 2 to
89/81/83 93, Pct 93, Pct 93, P c t
121 BP RRNA I to 9 5 , Mask 1 to 9 5 , Mask 1 to 95, Mask I to 95, Mask 4 homologies found.
UPDATED -2 t o -1 t o I to 2 to
e9/e1/83 93, P c t 93, Pct 93, Pct 93, Pct
W frm DNA f r o m DNA f r o m There were HAM45SRNA HAMMTRRl3 HMOSSRRNA HUMRR58S HUMRRSS DNA f r o m DNA f r o m DNA f r o m DNA f r o m There were MLYRRS
__-INFO
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ON
91.6, len 91.6, I e n 98.5, len
95. 95. 95.
188.8, l e n
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98.9, len 97.9, len 96.8, len
POSITx'dEs----
Ihumrr5s)
human,virus): What K r y w o r d s s h a l l I s r a r c h w l t h ? (r.g., S h a l l I s e a r c h f o r t i t l e c o d r s o n l y ( T ) or p r o v i d r d e t a i l r d l i s t i n g s (D):@ BIOCHEMISTRY-USA is, ses-sea ( 1 976) JOURWL
**********
LOCUS DE F IN IT ION REFERENCE TITLE AUTHORS JOURWL REFERENCE TITLE
HUlRRSS
H
121 BP
RRNA
UPDATED
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W 5s R I E O S W L RW. l 2 1 8 P
1 ( M S E S 1 TO 121) NUCLEOTIDE SEQUENCE OF KE CELL 55 RW FORGET,B.G. AND WEISSP14N,S.H. SCIENCE 158, 1695-1699 (1967) 2 (BCISES 1 TO 1 2 1 ) THE NUCLEOTIDE SEQUENCE OF R I B O S W L 5 S RIBCNUCLEIC ACID FRDH KE CELLS FORGET,B.G. AND WEISW,S.M. AUTHORS J B I O L CHEM 2 4 4 3148- 3165 ( 1 9 6 9 ) JOURNAL WrCT TO DO ANOTHER SEARCH? (IY/N)
:m
If YOU n r r d h r l p t y p r HELP. What k e y w o r d s s h a l l I s r r r c h w i t h ? : h a m r r g 5 s S h a l l 1 s e a r c h for t i t l r c o d e s o n l y ( T) o r p r o u i d e d e t a i l r d l i s t i n g s (D): WRRGSS SYRIAN MSTER 5s RIBOSWL RW GENE. 4 e e ~ p Do you w a n t a d r t r i l r d l i s t i n g for a n y o f t h r t i t l r s I f o u n d ( Y / N ) : n WfWT TO DO ANOTHER SEARCH? ( Y / N ) : Y
t
If YOU n r r d h e l p t y p r HELP. What K e y w o r d s s h a l l I s r a r c h w i t h ? (e.g., human,virus): f s h r r S s S h a l l I s e a r c h f o r t i t l r c o d r s o n l y ( T ) o r p r o v i d e d r t a i l e d l i s t i n g s (D): FSHRRSS TROUT (SFILMO GAIRDNERII) 5s RIBOSWL RW. i z e ~ p Do YOU w a n t a d r t a i l e d l i s t i n g f o r a n y o f t h r t i t l e s I f o u n d ( Y R J ) : n WfWT TO 00 ANOTHER SEARCH? ( Y / N > : n BEFORE YOU GO, w o u l d you I i k r t o r r v i r w p r o g r a m d r s c r i p t i o n ? : n
t
FIG. 20. (Continued)
method; the method has been applied to the design of DNA cloning experiments using a specific molecular biology knowledge base (Bach et al., 1984). A program has also been developed which can model a genetic regulatory system, the decision between lytic and lysogenic growth in bacteriophage A (Meyers and Friedland, 1984). These two projects indicate the potential of the application of expert systems for computer-aided experimental design and debugging, the building of molecular biology knowledge bases, and the modeling of biological systems, in applied genetic engineering environments.
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JOSEPH L. MODELEVSKY
Ill. Closing Remarks I have attempted to describe some of the major aspects of the design and application of complete computational support systems for genetic engineering. DNACE is an example of just one such system. Every computational system must be custom-designed to meet the needs of a specific user community. Not all user communities demand the same computational support. Not all user communities have the same list of projects and priorities. Therefore, no one system will likely be regarded as a complete system outside of its user community. Each computational system will be designed for particular hardware environments. The heterogeneous supply of hardware will ensure the heterogeneous development of software. The programming languages used for molecular biology software will also continue to be heterogeneous, and not necessarily portable. I look forward to the development of programming conventions in molecular biology computing. The implementation of BIONET, the national network for molecular biology computing, may establish a community which can formulate such conventions (Lewin, 1984). Perhaps workshops and meetings will be developed to provide the opportunity to organize this computing community. As I look forward (hopefully with “insight”), I expect that expert systems will ultimately be interfaced with automated data collection and analysis tools. These tools which assist the genetic engineer in data collection and reduction currently provide data which must be stored and forwarded by the scientist to other programs. The data could be compiled and passed to an expert system for evaluation and further instructions. Much genetic engineering technology is adaptable to automation. Automated processes can be computer controlled. Electronic workstations (technical assistants) that (1)monitor and control data-generating techniques, (2) collect data and then apply analyses like those detailed above, (3) expertly reduce the results, and (4) allow the scientist to rapidly develop conclusions, are tangible possibilities. The potential impact of such technology on science is well beyond the expertise of this author, but will, no doubt, provide the basis for many interesting discussions in the near future. ACKNOWLEDGMENTS The Eli Lilly and Company DNA Computing Environment (DNACE) is a result of a continuing collaboration between the Department of Scientific Information Systems and the Division of Molecular and Cell Biology Research of Lilly Research Laboratories. DNACE was initiated from discussions between Max Marsh and Richard Douthart. The DNACE applications pro-
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grams were expertly crafted by Franklin H. Norris, Gloria Griesinger, and Barry Stone. I thank John Wood for his critical review of this manuscript.
REFERENCES The Applications of Computers to Research on Nucleic Acids. (1982). Nucleic Acids Res. 10. The Applications of Computers to Research on Nucleic Acids 11. (1984a).Nucleic Acids Res. 12. Nucleotide Sequences (1984b). Part 2. Nucleic Acids Res. (Spec. Suppl.). Bach, R . , Iwasaki, Y., and Friedland P. (1984). Nucleic Acids Res. 12, 11-30. Buchanan, B. G. (1982). Department of Computer Science, Stanford University Report No. STAN-CS-82-953,pp. 1-13. Fickett, J. W. (1982). Nucleic Acids Rex 10(17), 5303-5318. Hopp, T. P., and Wood, K. R. (1983). Mol. Immunol. 20(4), 483-489. Kyte, J., and Doolittle, R. F. (1982)./. Mol. B i d . 157, 105-132. Lewin, R. (1984). Science 223, 1379-1380. Meyers, S., and Friedland, P. (1984). Nucleic Acids Res. 12, 1-10, Orcutt, B. C., George, D. G . , Fredrickson, J. A. and Dayhoff, M. 0. (1982).Nucleic Acids Res. 10, 157-174. Rindone, W. P., Merry, H. M., Goad, W. B., Bilofsky, H. S., and Carrico, C. K. (1983). Abstract. DNA 2, 173. Stone, B. N., Griesinger, G. L., and Modelevsky, J. L. (1984).Nucleic Acids Res. 12, 465-473. Zuker, M . , and Stiegler, P. (1981). Nucleic Acids Res. 9, 133-148.
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Reduction of Fading of Fluorescent Reaction Product for Microphotometric Quantitation G. L. PICCIOLO* AND D. S. KAPLAN**~,’ *Food and Drug Administration, Center for Deuices and Radiological Health, Rockville, Maryland, and +George Washington University, Washington, D.C.
.
I. Introduction . . . . . . . . . . . . . . . . . . . . . , . . , . . . . . . . , . . . . . . . . A. Scope of the Fading Problem B. Advantages of Reducing Fadin 11. Instrumentation . . . A. Macrofluorophot . . . ......................... B. Microfluorophotome B. Excitation Time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Environmental . . . . . . . . . . . . ... . IV. Comparison of Fading . . . . A. Excitation Source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Methods of Protection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Mechanism of Fading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Comparison of Protecting Agents . . . . . . . . . . . . . . . . . . . . . . . . A. Purpose .......................................... B. Materials and Methods . . . . . . . . . . , . . . . . . . . . . , . . . . . . . C. Results . . . . . . . . . . ....... D. Discussion ........................................ VI. Summary . . . . . . . . . . A. Progress in Redu B. Mechanism of Protection . . . . . . . . . . C. Improvements for Future Use . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . .
.
197 197 199 199 199 200 201 201 204 204 205 205 208 211 215 215 215 219 225 229 229 230 231 231
1. Introduction* A. SCOPEOF THE FADING PROBLEM Currently, evaluation of immunofluorescence (IF)3 test results is hampered by the rapid fading of the fluorescent reaction product (FRP)which, in ‘Submitted in partial fulfillment of Ph. D. requirements of the Department of Microbiology, George Washington University, Washington, D. C. 2Commercial equipment and instruments are identified by brand name and model in order to fully specify the experimental procedure. In no way does such identification imply recommendation or endorsement by the Food and Drug Administration, nor does it imply that the equipment identified is necessarily the best available for the purpose. 3Abbreviations: AO, acridine orange; ANA, antinuclear antibody; BSA, bovine serum al-
197 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 30 Copyright 0 1984 by Academic Press, Inc All rights of reproduction in any form reserved. ISBN 0-12-002630-9
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G . L. PICCIOLO A N D D. S . KAPLAN
TABLE I
METHODS FOR TILEREDUCTIONOF FADING Technique
Investigator
Localization under phase contrast Fast, epishutter excitation Chemical agents Pre- or postillumination Variable iris diaphragm on objective Neutral density filters Light sources Excitorharrier filters Field diaphragms
Ploem, Golden, Fukuda, Geyer Ploem, Golden, Geyer, Kaufinann, Nairn Gill, Johnsun, Sedat, Giloh, Kaplan, Picciolo Fukuda, Fujita Goldman, Ploem Nairn, Ploem Goldman, Haaijman, Johnson Goldman, Haaijman, Nairn, McKay Golden, Ploem, Haaijman
most cases, is fluorescein isothiocyanate (F1TC)-labeled antibody. Fading refers to the time-dependent decrease in fluorescence intensity upon continuous exposure to ultraviolet (UV) exciting light, as distinquished from quenching, which is a static reduction in the intensity due to some environmental or chemical condition present. Change in the fluorescence intensity with time is a measure of the fading rate, while the difference in the initial intensity under various environmental conditions is an evaluation of quenching. In the past, authors usually stated that fluorophores fade, and that researchers wanting to use the IF procedure would have to tolerate fading (Nairn et ul., 1969; Johnson et al., 1982; Schauenstein et al., 1975; Wick et aZ., 1975; McKay et al., 1981). IF tests are routinely used in clinical laboratories for serology testing and the IF-tagged reagents fade while the clinician is reading the slide. Newer methods applying quantitation of the intensity of the FRP emission also require that the FITC-conjugated reagents be stable during excitation. Several investigators have used various techniques to protect the sample from fading. These are summarized in Table I. Of these, the most promising is the use of chemical additives in the mounting medium that seem to protect the fluorophore from the effects of the excitation light. This article will discuss various methods that are reported in the literature and will bumin; CRT, cathode ray tube; CV, coefficient of variation; Dabco, 1,4-diazabicyclo[2.2.2]octane or triethylenediamine; DT, sodium dithionite or sodium hydrosulfite; DTE, dithioerythritol; DlT, dithiothreitol; FITC, fluorescein isothiocyanate; FRP, fluorescent reaction product; IgC, immunoglobulin G; IF, immiinofluorescence; NA, numerical aperture; nYG, n-propyl gallate or 3,4,5-trihydroxybenzoic acid n-propyl ester; PBA, pyrenebutyric acid; PBS, phosphate-buffered saline; PMT, photomultiplier tube; PPD, p-phenylenediamine; RB 200, rhodamine B 200; Tris, Trizma base or tris(hydroxymethy1)aminomethane; TRITC, tetramethylrhodamine isothiocyanate.
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present our data comparing the effectiveness of several additives in preventing fading under the same excitation conditions.
B. ADVANTAGESOF REDUCINGFADING It is anticipated that protection from fading would make exposure of the specimen to the exciting light less critical. This allows ease in the localization of the fluorescent specimens and permits more accurate discrimination between weakly positive and negative results, which is difficult if the sample is rapidly fading. Certain tests, such as determination of the type of herpesvirus present, require finding any positive cells that may be present on the entire slide. This searching procedure may take several minutes and must be done during excitation to recognize the presence of the positives. If fading is rapid, positives may be missed. Protection from fading is necessary in these cases. Reduction of fading would significantly improve quantitation of the FRP on IF microscopy slides. Retarding fading would permit longer scan times on slides without concomitant decreases in fluorescence intensity. This would permit the use of automated or semiautomated instrumentation which could scan a slide and determine the end point quantitatively. Rapidly fading specimens account for many false negatives in the clinical laboratory. In some cases, by the time the technician has set up the slide on the microscope, the weakly positive cell has faded to a negative cell. In the case of antinuclear antibody (ANA)-positive cells, the technician cannot properly identify the staining pattern if the specimen is rapidly fading. Stabilization of the fluorescence emission is necessary for objective, quantitative determination of antibody level.
II. Instrumentation Two general classes of instrumentation are available for intensity measurements of FRP.
A. MACROFLUOROPHOTOMETERS Macrofluorophotometers are designed to accept a cuvette that holds a volume of fluorescent solution or a solid sample holder that accommodates a flat plate. Both excitation and emission monochromators with variable slit widths are in the optical path (Sernetz and Thaer, 1973). A corrected emission spectrum is obtained using a standard emitter as a wavelength calibration. Macrofluorophotometers are effectively used for screening the effects of the chemical environment on fluorescence emission intensity, monitoring
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the purification procedures for the production of labeled conjugate, and determining the excitation and emission spectra of fluorophores. A detailed description of the Perkin-Elmer 650-40 spectrofluorophotometer and its use in predicting the effectiveness of reducing agents for protecting FITC-labeled conjugates from fading is given in Section V,B. Only part of the fluorophore solution in a cuvette placed in a macrofluorophotometer will be exposed to the excitation beam. The rest of the molecules are free to diffuse and effectively replenish the faded molecules, particularly if the solution is being mixed. Therefore, confirmation of the effectiveness of fading protection must be made in the microfluorophotometers.
B. MICHOFLUOROPHOTOMETERS Various types of microscopes are available with epifluorescence excitation and with a photometer to detect the emitted light intensity and to convert it
PMT DIAPHRAGMS REFLECTOR MERCURY BULB
OCULARS FIELD DIAPHRAGMS HEAT FILTER FILTER SETS EXCITER FILTER/ BARRIER FILTER
TUNGSTEN
OBJECTIVE SAMPLE -GLASS
..
LENS TRANSMITTED LIGHT SHUTTER
SLIDE
!
.*
'..*G-IRIS
DIAPHRAGM
FIG. 1. Schematic of light path for the Zonax microscope fluorophotometer.The epifluorescence exciting light (----), transmitted visible light (- - -), and the emission light (---) paths are shown.
REDUCTION OF FADING
20 1
to a digital signal. These systems have been described by others (Ploem, 1967, 1970, 1975, 1982; Ploem et al., 1974; Taylor and Heimer, 1974; Thaer, 1966; Golden and West, 1974). Figure 1 shows a typical epifluorescence optical path whereby the exciting light is incident through the objective on the specimen and the fluorescence emission is collected by the same objective and then focused on the photocathode surface of the photomultiplier tube (PMT). The advantage of epiilluminiation is the reduction of distortion of the emission spectrum due to reabsorption where there is an independence of section thickness. Therefore, one can measure surface fluorescence of opaque objects. There is also more precise alignment due to simultaneous focusing since the objective is the condensor (Rigler, 1966; Pearse and Rost, 1969). Several microscope systems are available with microprocessor control, including the operation of a 0.25- or 0.5-pm scanning stage. Leitz, Reichert, and Zeiss microscope companies offer such systems. We have used the Zeiss Zonax microscope and describe it in Section V,B.
Ill. Factors that Affect Fluorescence Intensity A. OPTICAL Comparison of fading results reported in the literature is difficult since the different parts of the optical system affect the degree of fading and the optical setup varied from one investigator to another. The following discussion summarizes each of the optical components that contributes to fading. 1. Lamp Housing
Depending on the design of the lamp housing of the excitation light source, the amount of light reflected to the collector lens will vary. Since fading is dependent on the excitation energy (Goldman, 1968; Haaijman, 1977; Lea and Ward, 1979) (see Section III,A,3), if excitation light is scattered in the lamp housing and lost through the baffles, a decrease in excitation energy occurs resulting in less fading (and less signal). The better designed lamp housings reflect a higher percentage of the excitation light to the collector lens. In addition, lamp housings vary in their efficiency of dissipating the heat from the light source. Heat buildup can cause instability of the light source (i.e., wandering of the arc or misfiring of the arc). An unstable light source causes fluctuation in the output from the lamp and will cause variations in the emission.
2. Light Source There are a variety of light sources available for fluorescence excitation. Lasers offer the advantage of delivering monochromatic light and can gener-
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G. L. PICCIOLO AND U . S . KAPLAN
ate pulses of light as short as 0.4 psec (Wick et al., 1975; Bergquist, 1973). These sources also give a high output of energy for exciting weakly fluorescent specimens. Additionally, lasers have a long lifetime compared to conventional light sources such as mercury, xenon, or halogen lamps. Mercury arc lamps emit strongly at several lines in the UV and blue light regions (365, 405, and 435 nm). Even though there is no special line in the spectral range of FITC absorption (440-490 nm), these sources are good for FITC emission (Goldman, 1968; Haaijman, 1977). Xenon bulbs produce a continuous emission throughout the entire spectrum but the brightness per unit area is lower than with mercury bulbs (Goldman, 1968). In addition, xenon lamps require the use of more restrictive filters than with mercury bulbs, since the excitation light continues into the emission region of the dye (due to the continuous spectrum). Xenon and mercry bulbs cause considerable fading of the specimen. Halogen lamps do not emit as much blue light and emit lower intensities than mercury lamps. Therefore, halogen lamps, in general, are not suitable for fluorescence quantitation (Goldman, 1968). These lamps are useful, however, when the specimen is brightly stained and the observer wants to eliminate fading as much as possible. Recently, IIBO 100-W mercury lamps have been developed with more stable arcs, more excitation energy, and less heat output energy. They are operated with a stabilized power supply and are currently the sources of choice.
3. Excitation Energy Ploem (1971)has shown that the fading rate is dependent on the excitation energy. Most researchers who perform experiments to measure fading have not measured the excitation energy of the light source, as the instrumentation to do this is specialized and expensive. The output varies from day to day and decreases as the bulb ages. Factors such as type of light source, age of bulb, position of the collector lens, diffusion of the light beam over the specimen, type of heat filters, magnification and numerical aperture (NA) of the objective, and type of excitation filters all affect the excitation energy. The fading of fluorescently stained specimens reported in the literature is not comparable unless related to the power density of the excitation light source. 4. Collector Lens
As light exits the lamp housing, the collector lens concentrates or diffuses it. The excitation energy is dependent on the position of the collector lens. If
the collector lens is adjusted so that the light is focused on a small spot on the specimen, then the energy per unit area will be higher than if the light is diffused over the entire field. Therefore, if the light is concentrated rather than diffused in order to increase the emission intensity, increased fading of the specimen will occur.
REDUCTION OF FADING
203
5. Heat Filters Heat filters are placed in the light path to filter out the infrared radiation so that the excitation filters are not cracked by the constant, intense radiation from the light source. In addition, these filters will decrease the transmission of light in the UV region to varying degrees, depending on the type and quality of the filter (Goldman, 1968).
6. Excitation and Neutral Density Filters
The amount and wavelength of the exciting light reaching the specimen are dependent on the filters used. Broad-band excitor filters allow a wider wavelength band to reach the specimen with more fading than narrow-band excitor filters. McKay et al. (1981) showed that using narrow-band FITC filters for blue light instead of UV blue reduced the fading and fluorescence intensity by equal amounts. Herzog et al. (1973) also found that the rate of fading is dependent on the filters used. Schauenstein et al. (1978) compared the excitation spectra of free FITC and conjugated FITC. They found that conjugation of protein to the FITC molecule quenches the UV maxima of 280 and 340 nm (the UV region) as compared to free FITC. Since there is no quenching at the 496 nm peak (the blue region), blue excitation is preferable when high intensities are desired. Ploem (1971) compared the fading of ANA-positive cells stained with FITC using various excitation filter combinations. The first combination (GG 475 and two KP 490 filters), which has a high transmittance (about 80%), showed a very rapid loss of intensity within 0.25 seconds. The second filter combination (the first with a 25% transmittance neutral density filter added) showed a much slower decay of the fluorescence intensity. Enerback and Johansson (1973) showed that inserting graded neutral density filters into the exciting light path proportionally reduced fading. Dichroic mirrors are interference-dividing plates that reflect light of certain wavelengths through the objective and allow light of shorter or longer wavelengths to pass through the filter, being lost through scattering (Ploem, 1970). Fading can be significantly enhanced or reduced depending on how selectively the dichroic mirrors filter out the light.
+
7 . Objectives Since in epifluorescence the objective acts as a condenser, the intensity of the light is dependent on the NA of the objective; the intensity increases as the square of the NA (Goldman, 1968; Haaijman, 1977). The NA is defined as the product of the refractive index of the medium in which the aperture angle is measured and the sine of the aperture angle (Piller, 1977). A typical NA for low-power objectives is 0.65, and 1.25 for high-power objectives. Some objectives have a variable iris diaphragm which allows the control of
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the excitation of the specimen. While reducing the excitation (via this method) does reduce fading, it does not allow absolute quantitation of the emitted intensity. Unless there is a very specific way of ensuring that the iris diaphragm is set to the exact same place each time, one cannot absolutely compare the intensities of samples. The type of objective will also influence fading. If the objective is made of several lenses which have been cemented together, there is approximately a 4% light loss each time the light passes through an air-glass interface (Zeiss, 1983). Depending on the number of lenses in the objective, this light loss could be significant if one is attempting to quantitate the fluorescence intensity. It should be noted that a similar light loss is observed in excitation filters which are composed of several filters cemented together.
B. EXCITATION TIME Not only is fading dependent on the excitation energy, but it is also dependent on the period of exposure. The longer a fluorescently tagged specimen is exposed to the exciting light, the more fading will occur, until a minimal plateau level is reached. Interspersing dark periods with excitation periods in some cases results in recovery of some of the intensity but this effect varies with fluorophore, exposure and dark times, and excitation energy. C. ENVIRONMENTAL In addition to fading caused by the optical elements, there are environmental factors which may affect fading. Haaijman (1977) compared fading of aminoethyl-Sephadex-bound FITC and Sepharose-bound FITC for 2 minutes under continuous excitation. FITC coupled to Sepharose faded 20% more than FITC coupled to aminoethyl-Sephadex beads. It is concluded that fading is dependent on the matrix to which FITC is bound. Haaijman (1977) tested the influence of pH on fading in the presence or absence of protein (i.e., CNBr-activated 4B-Sepharose-ovalbumin-FITC vs 4B-Sepharose-FITC) to test the hypothesis that electrophilic groups near the FITC moiety influence fading. Since fading in the presence or absence of protein was similar at various pH levels, he concluded that fading is not influenced by electrophilic centers in the protein to which it is coupled, but is a property of the molecule itself. McKay et al. (1981) found that when the pH of the buffered glycerol mounting medium was raised from 7.2 to 8.8, there was a 23% increase in the fluorescence intensity, but the rate of fading did not change.
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IV. Comparison of Fading
Since the above factors afTect the intensity of the emitted light, comparison of fading results from various optical setups in different laboratories is difficult. No set fading parameter has been established to allow this comparison. Therefore, the following fading percentages from various investigators are not directly comparable; however, an appreciation of the relative effectiveness of various conditions can be obtained. A. EXCITATION SOURCE
1. Laser Several authors have used lasers to measure fading of FITC-labeled conjugates (Wick et al., 1975; Kaufman et al., 1971; Bergquist, 1973; Bergquist and Nilsson, 1975; Schauenstein et al., 1980). These investigators compared fading of the conjugate when the sample was excited by repeated short pulses of light (using a laser) to fading when the sample was exposed to a conventional light source, such as a mercury or xenon arc lamp. Additionally, lasers have been used to measure recovery (the percentage of the initial fluorescence intensity that is regained as the cells are left in a dark environment) following various periods of fluorescence excitation. Experiments combining fading and recovery effects have been useful in explaining the mechanism of fluorescence fading. a. Argon Ion Laser. Kaufman et al. (1971) measured fading of FITClabeled Escherichia coli cells using an argon ion laser. At a power density of 160 W/cmZ, 89% of the initial fluorescence intensity faded within 10 seconds, under continuous irradiation. However, when the excitation time was reduced to milliseconds, no significant fading could be detected. Schauenstein et al. (1982) found that free FITC in solution lost 40% of its initial intensity during the first 100 msec of excitation with an argon laser.
b. Pulsed Dye Laser. Bergquist and Nilsson (1975) compared fading of FITC-labeled, glutaraldehyde-polymerized microspheres of purified human immunoglobulin G (IgG) excited with an HBO 200-W mercury lamp to fading when the spheres were excited with a Chromabeam 1070 pulsed dye laser. The laser was adjusted to produce light of 495 nm. Bergquist (1973) has previously shown that when the spheres were exposed to a total of 125 pulses (each pulse is 0.4 psec for a total exposure time of 50 psec) and the resultant image was exposed to photographic emulsion, there were no signs of significant fading. In a second study (Bergquist and Nilsson, 1975), they
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repeated the previous study and quantitatively measured fading by monitoring the deflections on an oscilloscope from the photomultiplier tube (PMT). They found that even after 50 laser pulses had illuminated an individual sphere, no fading was observed. However, after 1 second of exposure to an HBO 200-W mercury light, only 85% of the initial intensity remained.
c. Recove y. Since researchers and laboratory technicians are usually interested in observing a fluorescently stained field more than once (i.e., in the histopathological diagnosis of cancer cells or when observing the staining pattern in immunofluorescence diagnostic test kits), researchers are interested in determining the extent of permanent lowering of the fluorescence intensity by prior excitation conditions. Maintaining the level of the initial intensity is important in the diagnosis of disease states since it is often necessary to have a second technician or a doctor review the test results. If the fluorescent field is irreversibly faded during the initial observation, then confirmation of the first technician’s diagnosis is impossible and falsepositive or false-negative results may be reported. Kaufman et al. (1971), Wick et al. (1975), and Schauenstein et al. (1975) found that recovery is dependent on the time of exposure to the excitation light source and the length of time the specimen is left in the dark following excitation. These authors found that a minimum dark period of 2 seconds between laser pulses is necessary for recovery of fluorescence. Schauenstein et al. (1982) found a 60%recovery using two pulses of 3 msec each with a 3 msec dark interval. However, in many cases, recovery is partial or does not occur at all. Recovery is negatively related to the product of the excitation time and intensity of the exciting light and is positively related to the time the sample is left in the dark following excitation (Wick et al., 1975; Kauffman et al., 1971; Schauenstein et al., 1975).
2. Conventional Light Sources Most fluorescence microscopes (i.e., those used in hospital or research laboratories) are equipped with either a halogen, mercury, or xenon light source. The average hospital laboratory cannot afford a laser excitation source nor does it have the personnel qualified to properly align the light source. In addition, laser light sources require the use of special low-fluorescence optics and filters to avoid autofluorescence of the optical system (Kaufman, 1971). a. Mercury Lamps, Nairn et al. (1969) measured fading of rat gastric cells stained with FITC-conjugated antihuman globulin using an HBO 200W mercury lamp. When the specimen was mounted in buffered glycerol at pH 8.6 and excited with only ultraviolet light, 35 seconds was required to
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fade half the initial intensity. When the sample was excited with UV blue light, the half-life decreased to 4 seconds. After 1 minute of continuous excitation with UV irradiation, only 30% of the initial intensity remained. Haaijman (1977) compared fading of aminoethyl-Sephadex-bound TRITC (tetramethylrhodamine isothiocyanate) and membrane-bound TRITC. Membrane-bound TRITC faded about 25% more in 2 minutes than the TRITC bound to the beads. He found the FITC and TRITC bound to aminoethyl-Sephadex beads faded less rapidly than cell-bound conjugates. Golden and West (1974) measured fading of Ehrlich’s hyperdiploid, mouse ascites tumor cells stained with acridine orange using an HBO 100-W mercury lamp. They describe fading in terms of a time constant, 7 , which is approximately 1.8 seconds. Although not specifically stated, it can be inferred from the fading curve that T is the time required to fade to 37% of the initial intensity. These data show that fading can be approximated with a single exponential. The shape of this fading curve is dependent on cell type and substrate biopolymer.
b. Xenon Lamps. Using an XBO 75-W xenon lamp, McKay et al. (1981) measured fading of conjugates of antihuman y-globulin-FITC and antihuman y-globulin-rhodamine B 200 (RB 200). With the RB 200 conjugates, there was little if any fading after 2 minutes, and this decline could not be separated from instrument error. For fluorescein, however, there was considerable fading which reached a plateau after a certain period of time. This result was interpreted to mean that fading is the sum of two components, one that decays exponentially and one that remains constant. They subtracted the plateau level value, which represents the nonfading component, from each intensity value and plotted the fading component vs time on semilogarithmic paper. This plot produced a straight line which showed that fading obeyed first-order kinetics. They found a half-life of about 1 minute for their FITC conjugates. Enerback and Johansson (1973) measured fading of several fluorochromes including FITC and Feulgen-Schiff using an XBO 75-W xenon lamp and instrumentation capable of recording fluorescence of very short duration. They found a half-life of 2 seconds for FITC under continuous excitation. For the Feulgen-p-rosaniline reaction, there was a 20% loss of initial fluorescence after 20 seconds. They also tested the effect of repeated very short excitation times at 2 second intervals on fading. For FITC, there was significant fading after 15 measurements with illumination times up to 1/60second. Using an oscilloscope, 0.5%fading occurred during the first 2 msec of illumination. For Feulgen-Schiff-stained cells, fading could be prevented by reducing the illumination time. Bohm and Sprenger (1968) measured fading of sperm stained with several dyes, including acriflavine and p-rosaniline under 5 minutes of continuous excitation using
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an XBO 150-W xenon lamp. They found a fading rate of 60 and 25%, respectively.
c. Recovery. McKay et al. (1981) tested the recovery of FITC-stained cells using a 75-W xenon bulb. They allowed the FITC conjugate to fade approximately 6 half-lives and then measured the intensity by varying the dark period between excitations using an excitation shutter. They found that if the shutter was opened for only 3 seconds every 5 minutes, the intensity increased from 64 to 99 (33%).This recovery was probably real because it could not be obtained on unstained specimens. These data correlated with the recovery experiments performed using lasers. B. METHODSOF PROTECTION To improve the accuracy of the IF test and allow quantitation of the fluorescently emitted light (since quantitation is not feasible on fading specimens), researchers have tried various ways of stabilizing the fluorescence emission (Table I).
1. Chemical Agents When selecting possible chemical agents to retard fading, it is important that the agents do not fluoresce at or near the excitation or emission wavelengths of the dye. Q. Sodium Dithionite. Gill (1979) used sodium dithionite (DT) to inhibit the fading of onion cuticle cells labeled with fluorochromes such as fluorescein, acridine orange, Hoechst 33258, acriflavine, and others under continuous excitation for 2 minutes with an HBO 200-W mercury bulb. It should be noted that he did not use these dyes conjugated to antibodies. He found that for fluorescein and acridine orange, the intensity increased before starting to decrease after 5 minutes of continuous excitation. Gill’s data showed that after normalizing the intensities, the ratio of the intensity at 2 minutes of excitation to the initial intensity was 0.67 for the buffer control and 1.00 for the mounting medium with DT.
b. n-Propyl Gallate. Giloh and Sedat (1982) incorporated n-propyl gallate (nPG) into the mounting medium to retard fading during serial photographs of nuclei of fixed, cultured Drosophila cells incubated with a monoclonal antibody against Drosophila melanogaster embryo nuclei. They found that 2-5% nPG in glycerol reduced fading of tetramethyl rhodamine isothiocyanate (TRITC) and FITC by a factor of 16 and 7 times, respectively. At concentrations of 10-20% nPG in glycerol, self-quenching occurs. They also
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noted that free radical scavengers such as dithiothreitol (DTZT) at concentrations of 0.05-0.2 M in 90% glycerol had no effect on fading. This is in contrast to our results with dithioerythritol (DTE) or DTT as shown in Section V,C,5. Giloh and Sedat advise that the initial fluorescence intensity may decrease upon storage in nPG. The decrease in intensity can be reversed or prevented by washing the slides in phosphate-buffered saline (PBS) and storing them in pure glycerol.
c. p-Phenylenediamine (PPD) and 1,4-Diazabicyclo[2.2.2]octane (Dabco). Johnson et al. (1982) and Johnson and de C. Nogueira Araujo (1981) added PPD or Dabco to the buffered glycerol mounting medium to reduce fading during examination of cells for ANA staining. Using a 16X Planachromat objective and PPD at a concentration of 0.01 M , they found that about 90%of the initial fluorescence intensity remained after 5 minutes of continuous excitation with an HBO 50-W mercury lamp. Dabco provided similar protection when used at a higher concentration of.0.2 M. When the magnification was increased to 40X10.95, with both PPD and D,,, about 60% of the initial intensity remained after 5 minutes of continuous excitation. For the glycerol controls using the 40X 10.95 objective, only 10-20% of the initial intensity remained after 5 minutes of continuous excitation. The authors recommend Dabco over PPD since the latter is a skin sensitizer, is photosensitive, and undergoes oxidative degradation. They also compared fading of stained nuclei in the presence or absence of protecting agents using an HBO 50-W mercury and an HBO 100-W mercury lamp (incident illumination) and a quartz-iodine (QI) lamp (transmitted, darkfield illumination). The relative initial fluorescence intensities of the three lamps were 1 2 5 1 , respectively, for HBO 100:HBO 50:QI. At low magnification (16x), fading was similar for all three lamps. An important point to note is that blank readings (unstained sections mounted in the same medium as the stained slide) accounted for as much as 25% of the readings on stained cells. The blank readings were subtracted from the corresponding reading from the stained sections. Johnson et al. postulate that the blank reading accounts for the nonfading component described by McKay (1981). However, the blank readings that McKay used were on stained slides from an area of nonspecific staining and his values were much lower than 25%. Whether it is valid to use unstained cell emission as a background for stained cells is unclear. No data were given by Johnson et al. to document that this is a true reflection of the fluorescence intensity that the stained cells emit nonspecifically. This is particularily important since the value is so large relative to the specific intensity, and because the counterstain is added to mask nonspecific intensities. The counterstain emission itself is excluded by the filter selection.
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2. Fixation in a Nonjluorescent Resin As described below, mounting the specimen in a nonfluorescent resin reduces fading by stabilizing the macromolecule-dye complex. Rodriguez and Deinhardt (1960) used polyvinyl alcohol to prepare semipermanent mounting medium and to reduce fading upon storage. They found that slides stored at 4°C and frequently exposed to room temperature for hours at a time did not show appreciable fading for periods exceeding 9 months. Fukuda et al. (1980) stained smears of mouse hepatocytes with an anti-UV DNA antibody and an FITC-labeled antibody and measured the fading in glycerin or buffer with methanol postfixation. After 20 minutes of continuous excitation, the fluorescence was nearly immeasurable. However, postfixation of the specimen with absolute methanol for 1 hour followed by mounting in a nonfluorescent resin greatly reduced fading. The mechanism active here is most likely the removal of water with its component of dissolved oxygen. This provides more rigidity to the fluorophore complex and less opportunity to interact with oxygen which accelerates the fading rate. They found no detectable fading after storing the specimens for 2 years at room temperature without shielding against light. McKay et al. (1981) found that by mounting the specimen in pure glycerol or butanol instead of 15% glycerol, the quantum yield of RB 200 in solution could be doubled and the intensity of fluorescence of stained slides was increased by almost 50%. Again, this is consistent with the above mentioned mechanism of decreased water concentration providing more efficient fading protection, since alcohol is a dehydrating agent.
3. Pre- or Postfixation of Specimen Fukuda and Fujita (Fukuda et aZ., 1975, 1976, 1977; Fujita, 1973; Fujita and Fukuda, 1974) used another method to eliminate fluorescence fading of Feulgen-stained nuclei. They either pre- or postirradiated (after nuclear staining) the specimen for up to 20 hours to selectively remove nonspecific fluorescence and subsequently the stain retained the proportionality between DNA content and stain concentration. This method is based on the fact that nonspecific fluorescence decays faster than specific fluorescence. Therefore, by carefully adjusting the pre- or postillumination time, one can selectively remove the unwanted fluorescence and not destroy the specific fluorescence. Fukuda postfixed a pyrimidine dimer-FITC complex with ethanol and mounted the specimen in Entellan (a nonfluorescent mounting resin). The specimens were irradiated with violet light for FITC (405 nm), before or after staining, for 5 hours. They found that postirradiation of the specimen with violet light for appropriate times after staining reduced background fluorescence and decreased fading of tissue-bound FITC. Fukuda
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standardized the conditions for postirradiation for DNA cytofluorometry on a Feulgen-p-rosaniline-stained smear. They found that a postirradiation of 10 hours retained proportionality between DNA amount and fluorescence intensity. This method essentially accelerates fading to a plateau level and thus provides minimal subsequent fading so that a more stable emission measurement is obtained.
C. MECHANISMOF FADING
The exact mechanism of fluorescence fading is not known. Many papers have been published on possible mechanisms; these mechanisms are based on data obtained by performing studies such as recovery experiments, flash photolysis studies, solution fluorescence in the presence or absence of molecular oxygen, and comparison of fading in distilled water vs deuterated water. It is commonly believed that fading is due to the reaction of the excited electrons of the fluorochrome with oxygen to form an oxidized nonfluorescent by-product (Menter et al., 1978, 1979; Giloh and Sedat, 1982; Vaughan and Weber, 1970). 1. Principle of Fluorescence and Phosphorescence
The following discussion of fluorescence and the discussion of the fate of the excited electrons are according to McCarthy and Moyer (1970). Upon excitation with ultraviolet light, the electrons of the fluorophore are excited from the ground state to a higher energy level. In the ground state, the orbitals contain two spin-paired electrons (i.e., the spin orientation is in opposite directions). The net spin in the ground state is zero. By the absorption of energy from the exciting light, the electron may be promoted from the ground state orbital to one of the excited state orbitals. The excited state orbitals possess more energy than the ground state molecules. The net spin of the excited electron state will be the same as the ground state. To note this similarity, the multiplicity of the ground and the excited state is calculated by means of the formula M = 2s 1, where M = multiplicity and S = spin. States with a multiplicity of 1 are designated singlet states. There are many levels of excited singlet states, and these are assigned ordinal numbers to designate their relative energy. It is also possible for the promoted electron to reverse its spin when it goes to the excited state, causing a net spin of one. The multiplicity is then equal to three. This state is designated the triplet state. Molecules with net spins greater than zero are paramagnetic. An important point to note is that the energy of a triplet state is always lower than the energy of its corresponding singlet state. Again, there are many triplet states for a given molecule. The lowest energy triplet state corre-
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sponds to the first excited singlet state and not to the ground singlet state. The energy necessary for transitions between two electronic states is a continuum so that any energy supplied to the molecule between two boundaries can cause the promotion of electrons to a higher state. Once the electrons have been promoted to the excited state, there are many different reactions that can occur. (1)Initial deactivation involves the rapid production of molecules in the lowest vibrational level of the first excited singlet state, S,. This process is usually radiationless. (2) Fluorescence is the radiational deactivation from S, to So. (3)Quenching involves interaction of the S, molecule with the environment resulting in a nonradiational deactivation. (4) Radiational deactivation from T, to So is phosphorescence. (5)Intersystem crossing involves the flip of an electron spin. These types of interactions are, in general, considered quantum mechanically forbidden, but there is a finite probability of intersystem crossing for many molecules. (6)There can be TI to S, transitions. Slow (delayed) fluorescence is the radiational deactivation of S, to So after a T, to S, IC. For additional sources on the principles of fluorescence, the reader is directed to Udenfriend (1962), Goldman (1968), Piller (1977), and Guilbault (1967).
2 . Recove y Bergquist (1973) postulates that fluorescent dyes are protected from intense irradiation when they are bound to large molecules such as IgG and that fading occurs stepwise as dye is released and decomposes. Therefore, since no fading is observed when very short exposures with laser light are used, then longer times are needed for the reactions which cause fading. Kaufman et al. (1971) speculate that recovery is due to recombination of the dye and conjugate over a period of time. Wick et al. (1975) and Schauenstein et al. (1975, 1982) postulate that fading involves two processes occurring in parallel, i.e. (1)the reversible relaxation of the electron singlet state (which is very sensitive to changes in the chemical and physical microenvironment of the dye molecule), and (2) the irreversible dye decomposition caused by nonradiative transitions. They theorize that recovery is induced by the formation of the triplet state, which is dependent on the excitation energy. The triplet states decay and form singlet states which decay to the ground state with the emission of fluorescent light.
3. Schi-ff-Type Dyes Fukuda et al. (1980) evaluated factors that affect fluorescence fading in cytofluorometry. Using fluorochromes such as Feulgen-p-rosaniline, Hoechst 33258, acriflavine-SO,, and cresyl violet-SO,, the authors tested the effect of treating the specimens with RNase, trypsin, or hypotonic solution before staining. The effect of mounting the specimen in a nonfluores-
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cent resin after poststaining fixation with absolute methanol was also tested. They found that postfixation with methanol and mounting in a nonfluorescent resin greatly stabilized the fluorescence emission. Fukuda et al. also found that the conformational protection of the macromolecule-dye complex is important in fading and that an increase in conformational flexibility of dye-binding macromolecules, caused by treatment with factors such as RNase, trypsin, or hypotonic solution, increases fluorescence fading by allowing increased photochemical reactions and interactions of the fluorochrome. Factors which increase the rigidity (i.e., mounting in nonfluorescent resins) of the dye-binding macromolecules decrease fading. Therefore, changing the p H of the mounting medium and heating up the slide by the excitation light cause instability of the immunoglobulin-dye complex and increased fading.
4 . Acridine Orange Menter et al. (1978, 1979) used complexes of acridine orange (A0)heparin in solution to elucidate the mechanism of fading. They photolysed complexes of acridine orange-heparin in N2-, 02-, or air-saturated solutions. They found that the 02-saturated solutions faded seven times faster than the N2-saturated solutions. They postulate that the fading of A 0 absorbed to heparin is a special case of dye-sensitized photooxidation of tertiary amines in which bound A 0 acts both as a sensitizer and substrate amine. Fading involves the intermolecular transfer between adjacently bound excited and unexcited dye. Electron transfer could be the rate-limiting step in fading. Photolysis of the complex results in irreversible photooxidation of the dye. They postulate that 0, provides additional pathways for by-products and prevents back reaction between radicals to form ground state molecules. Bellin (1968) discusses the properties of dyes bound to polymeric substrates. These dyes included acriflavine, eosin, fluorescein, and triphenylmethane. She states that the binding of dyes to polymeric substrates increases the population of the triplet state and the chance of photoreduction and that the quantum yield of photoreduction is proportional to the concentration of bound dye molecules. On the other hand, dye binding to polymers usually protects the dye from photooxidation. Bellin concludes that dye binding increases the formation of the triplet state and the susceptibility to photoreduction, which is proportional to the concentration of bound dye; and it also increases the ability of the dye to act as a photosensitizer in photoreduction.
5 . Pyrenebutyric Acid Vaughan and Weber (1970)measured the quenching of pyrenebutyric acid
(PBA) solutions as a function of the oxygen concentration in the solution and
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the conjugation of PBA to various proteins. They found that bubbling oxygen through a dilute solution of PBA in water decreased the fluorescence lifetime of free PBA from 100 to 65 nsec. This quenching was proportional to the absolute temperature and inversely proportional to the viscosity of the solution, which means that fading is primarily due to collisions between 0, and PBA. Oxygen was 99.5 h 10% efficient at quenching the fluorescence of PBA. Since quenching is primarily due to oxygen diffusing through the solution, they covalently conjugated PBA to bovine serum albumin (BSA) to reduce the availability of PBA to 0,. In this case, the fluorescence lifetime was increased from 139 (free PBA) to 172-204 nsec (conjugated PBA). When the BSA was denatured in 8.6 M urea, the lifetimes of the free and conjugated BSA became practically identical. Vaughan and Weber also showed that as the water content of a glycerol-water mixture is increased (after bubbling with N, for 10 minutes), the fluorescence lifetime decreases. Additionally, the absorption maximum decreases from 345.5 to 342 nm. These investigators concluded that oxygen quenching of PBA depends on the rate of emission of the fluorophore and on contact with free-diffusing oxygen. 6. FlTC
Johnson et al. (1982)reported test results which implied that oxygen is not involved in the fading of FITC-conjugated antibodies. They used protecting agents such as Dabco and PPD to retard the fading of ANA-positive cells stained with antihuman IgG-FITC conjugate. Their results were as follows: (1) the FITC conjugate in PBS with oxygen faded faster than the conjugate in PBS with argon (55 vs 30%, respectively, in 30 minutes); (2) fading of solutions of FITC in oxygenated distilled water and fading in oxygenated deuterium oxide were similar, i.e., no solvent isotope effect; (3) using glycerol solutions containing buffered FITC conjugates, fading was less rapid and seemed to be independent of the presence of oxygen; and (4) flash photolysis studies showed that Dabco, NaI, or NaN, does not quench the triplet state of fluorescein, but do quench the excited singlet state. The isotope experiments suggest that singlet oxygen is not involved in the fading mechanism. This conclusion is further supported by the glycerol experiments which showed a similar fading rate in the presence or absence of oxygen. Agents such as Dabco, NaI, and NaN, are capable of inhibiting the reaction with singlet oxygen, and quenching the excited singlet or the excited triplet state. Since the flash photolysis studies showed that these agents do not affect the triplet state and the lack of a solvent isotope effect ruled out the involvement of singlet oxygen, they concluded that their data prove that reducing agents suppress a destructive reaction of the dye in the excited singlet state with protein. In contrast, Giloh and Sedat (1982) postulate that since compounds
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such as nPG, ascorbic acid, PPD, and dithionite had a similar protecting effect on fluorescein, molecular oxygen is involved in fading reactions.
V. Comparison of Protecting Agents A. PURPOSE Because of the difficulty of comparing reports on the effectiveness of protecting agents performed under various excitation and measurement conditions, we tested the protective effects of several chemical reducing agents under the same excitation conditions. The agents compared were DT, D I T , DTE, and Dabco. We did this using a macrofluorophotometer for screening effectiveness and a microfluorophotometer for verifying “in use” conditions. The kinetics of the fading curves were analyzed, and implications for elucidation of the mechanisms of fading and protection are presented.
B. MATERIALS A N D METHODS
1. Instrumentation a. Zonax. A Zeiss microscope-photometer is adapted for epifluorescence (incident excitation) using an HBO 100-W mercury lamp with a stabilized DC power supply. Figure 1 shows a schematic of the light path. A microprocessor, Zonax, is integrated with the microscope. A wide-band FITC filter set (Zeiss, Product No. 487709) was used: excitation, 450-490 nm; dichroic mirror at 510 nm; and barrier filter at 520 nm. The filters were mounted in the Zeiss III-RS illuminator filter holder which contained positions for four combinations. A heat filter (Heat reflecting Calflex, Zeiss, Product No. 467832) placed in the exciting path minimizes intensities from background materials and reduces fading. A linear interference monochromator is placed in the emission light path to the PMT. Attached to the monochromator on the microscope is a Hamamatsu PMT (type R928 multialkali photocathode, 9 stage, side-on), powered by a stabilized high-voltage power supply. An amplifier was built into the PMT housing. The emitted intensity was converted into a voltage displayed on the computer cathode ray tube (CRT) screen. A series of variable field stops can mask down the area of the specimen actually illuminated by the exciting light. Field stops ranged from 0.05 to 2.5 mm diameter. Adjacent to the PMT, in the emission light path, are diaphragms that can vary the area being measured. Diaphragms ranged from 0.08 to 5 mmmdiameter. The amount of fading during measurement can be reduced by a fast shutter (msec). We
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used a 6 3 x f 1 . 4 planapo objective. The microscope is equipped for brightfield, darkfield, and phase contrast for localizing the specimen. Software programs (provided by Zeiss) control the microscope shutters, field stop, PMT diaphragm, high voltage, gain, and the scanning stage. A measurement protocol, either automatic or manual, may be used as well.
b. Zonax Calibration. Fluorescent materials available for use as calibrators of fluorescence instrumentation were surveyed and tested. These results will be reported elsewhere (Kaplan and Picciolo, 1984). One of the most reliable of these was the uranyl plate (Corning, Product No. 3718). By special order, a modification of this uranyl plate was provided in the shape of a microscope slide. The slide was found to be nonfading and thus was used to evaluate the stability of the microscope-photometer. Under continuous excitation for 3 hours, a negligible slope of -0.008% per second for the linear regression line and a coefficient of variation of 0.313%were obtained. Using uranyl glass slides with the optimal field stop and PMT diaphragms, we determined fluorescence intensity values using all possible combinations of amplifier gain and high-voltage settings which result in measureable intensities. Multiplying the readings by the gain settings of the PMT amplifier for each high-voltage setting corrected the readings for various gain settings. The corrected intensity-voltage relationship was linear on a log-log plot. This indicates that the intensity is related to the high voltage as a power function, as is expected. The equation for this was PMT output = (10-19.34)(high v ~ l t a g e ) ~ . ~ To compare intensity readings day to day, we read the uranyl glass slides before andlor after each experiment. Since the uranyl slides contain nothing upon which to focus, we adjusted the focus knob until we obtained the highest intensity reading. The maximum intensity was constant across approximately one-half of a turn of the fine focus knob, indicating that the focal level on the uranyl glass slide is not critical within this amount and allowing confidence in the readings. Since the uranyl slide contains fluorophore throughout its entire thickness, it is assumed that the focal level of maximum intensity represents the level at which the focal cone is filled with fluorescence from a solid angle relative to the numerical aperture of the objective used. An alternative method has been suggested, that is, to scratch with a diamond point a mark on the surface of the glass and to focus on this. This however, is not satisfactory for two.reasons: (1)the scratch is hard to find and often disappears when oil of a certain refractive index is added to the slide, and (2) the focal level is at the surface of the slide and since there is no fluorophore above the surface, small variations in focus will introduce large
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variations in the amount of light measured. The initial reading of the uranyl glass slide was set to 100 to ease the mathematical manipulation and to make full use of the graphics screen on the CRT. The high voltage used was 517 V and the amplifier gain was one. All further readings of both standards and samples were made at high-voltage and gain settings that gave relative intensity values close to 100. Then these high-voltage and gain values were used to correct the intensity readings of the sample relative to the uranyl glass slide reading for the day. This was done using the regression line parameters to calculate the extrapolated intensity at the standard settings of 517 and one.
c. Perkin-Elmer Calibration. The Perkin-Elmer 650-40 spectrophotofluorometer is microprocessor controlled and includes software to correct the sample fluorescence spectrum by reference to the emission spectrum of RB 200. This is actuated by setting the corrected mode after running the RB 200 spectrum. The fluorometer uses a second photodiode to automatically correct the dynode voltage for fluctuations caused by the light source which is the 150-W xenon lamp with stabilized power supply. This is actuated by setting the ratio mode. The fluorescence intensity readings are displayed in digital form on the fluorometer display. Another software option allows repeated scanning of the fluorescent specimen between preselected wavelengths and an average curve to be drawn from the individual curves.
2. Reagents a. Reducing Agents. The chemical reducing agents tested were sodium dithionite (DT, sodium hydrosulfite), Aldrich Chemical Company, Milwaukee, Wisconsin (Catalog No. 15,795-3); dithiothreitol (D'IT), Sigma Chemical Company, St. Louis, Missouri (Catalog No. D0632); dithioerythritol (DTE), Sigma Chemical Company (Catalog No. D8255); Dabco (1,4-diazabicyclo[2.2.2]octane),Aldrich Chemical Company (Catalog No. P3130). DT, DTT, and DTE were prepared as stock solutions containing 0.5 M reducing agent in 0.5 M tris(hydroxymethy1)aminomethane (Tris) buffer, p H 8.2 (Trizma base, Sigma Chemical Company, Catalog No. T-1503). Stock solutions were aliquoted and frozen for future use to preserve the potency of this material. For use in the Perkin-Elmer macrofluorophotometer, doubling dilutions of the reducing agents were prepared in the concentration range 0.063-0.5 M in 0.05 M Tris, pH 8.2. The diluted reducing agent was then diluted, one part reducing agent to nine parts of a mixture of 0.05 M Tris, pH 8.2, and FITC-labeled conjugate. For experiments using the Zonax microscope, dilutions in the range 0.25-0.5 M were prepared. One part of the concentrated reducing agent was added to nine parts buffered glycerol mounting medium.
218
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L. PICCIOLO A N D D. S. KAPLAN
For Dabco, doubling dilutions at a concentration range of 0.03-0.5 M were prepared in 0.5 M Tris, p H 8.2, for determination of the optimal concentration and a final concentration of 0.3 M Dabco in buffered glycerol was obtained by diluting a stock solution for the other experiments. nPG would not go into solution at a concentration of 2-5% as used by Giloh and Sedat (1982). Therefore, we did no further experimentation with this material.
b. Conjugates. The following FITC-labeled conjugates were used: (1) goat antihuman polyvalent globulin to rubella virus with rhodamine counterstain incorporated [Electronucleonics, Inc. (ENI), Columbia, Maryland]; (2) goat antihuman polyvalent globulin to ANA with rhodamine counterstain incorporated (ENI); (3) goat antihuman IgG (heavy and light chains) to T o x o p l a m gondii with Evans blue counterstain (ENI); (4) goat antihuman polyvalent globulin for T . gondii without counterstain incorporated (Center for Disease Control, Atlanta, Georgia); (5) rabbit antihuman (IgG) globulin to Neisseria gonorrhoeae with and without rhodamine counterstain. 3. Methods a. Measurement of Fading in Macrofluorophotometer. The fading of FITC-labeled conjugates (without added cells) with varying concentrations of reducing agent was measured in the Perkin-Elmer fluorometer and compared to controls. The following conjugates were measured: T . gondii (without counterstain) and N . gonorrhoeae (with and without rhodamine counterstain). The excitation and emission wavelengths used were 498 and 522 nm, respectively. The slit widths for excitation and emission (respectively) were 20 and 5 nm. The samples were continuously excited with a 150-W xenon light for 10 minutes and intensities integrated for 15 second intervals using the corrected spectrum option. From these data a plot of corrected intensity vs time was prepared.
b. Measurement of Fading in Microjluorophotometer. Measurement of fading in the Zonax microscope was done using the kits for ANA, rubella virus, and Toroplasma. The I F microscopy slides from commercially available kits were prepared according to each manufacturer’s directions except that an optimum concentration of reducing agent was incorporated into the buffered glycerol mounting medium provided with the kit just prior to mounting the slides. Whenever possible, the cells were located under transmitted visible light in order not to fade the specimen. The high voltage and the amplifier gain to the PMT were adjusted so that the initial intensity would be 100%.The sample was continuously exposed to excitation light using a wide-band FITC filter combination (Zeiss, Product No. 487709) and
REDUCTION OF FADING
219
intensity measurements were automatically taken every 0.1115 minute for 10 minutes using the kinetics software. A KP-560 bandpass filter was placed in the emission path to eliminate red emission light. The fluorescence intensities were later corrected to a standard high voltage and gain, based on the statistical regression parameters of the uranyl glass slide used to calibrate the instrument daily, to allow direct comparison of cell intensities independent of high-voltage and gain settings and daily lamp fluctuations. Background readings were taken using the same filter combination as for the samples by measuring an adjacent area of the stained tissue or cells that showed the nonspecific staining using the same diaphragm areas. The background readings were usually less than 1%of the sample readings. The background readings were subtracted from readings of the specific intensities. c. Statistical Analyses. One of the software packages available with the Zonax allows the generation of kinetics graphs (a graph of intensity over a user-predetermined time frame). The kinetics plot allows one to look directly at the percentage fading of the sample. The software also calculates the coefficient of variation (CV) which allows comparison of the fading of the cells independent of the mean. Use of a data link between the Zonax and an IBM host computer allowed generation of a variety of statistical analyses including regression, ANOVA, and graphics output from the original data generated by the microscope.
C. RESULTS 1 . Selections Criteria
DT, DTT, DTE, and Dabco were tested to determine if they could effectively protect the FITC-labeled cells from fading. These agents were evaluated on the basis of five criteria: (1)effective protection of the sample from fading; (2) no inhibition of the initial fluorescence intensity of the fluorophore; (3) no increase in the background fluorescence; (4) ability to function with the buffer, pH, molarity, and temperature used with the mounting medium in the fluorescence test kits; (5) practicality. The Perkin-Elmer spectrofluorophotometerwas used to screen the reducing agents for their protective ability. The fluorometer has the advantage of allowing rapid screening of the prospective agents without requiring several hours to prepare I F microscopy slides. 2. Buffer Type
Chemicals capable of buffering in the pH range 8.0-9.0 were selected. Glycine adequately buffers in this range but has a high background intensity
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(autofluorescence). Tris buffer maintains the pH within the desired range and does not significantly autofluoresce at the excitation and emission wavelengths used. PBS did not adequately buffer the reducing agent solution and was, therefore, not used further.
3. Buffer Concentration Experiments were performed to determine the lowest concentration of Tris buffer that was still capable of maintaining the pH of the reducing agent at 8.2. A 2.0 M stock solution of D'IT was diluted in Tris buffer solution to concentrations between 0.2 and 0.02 M and the pH of the solution was measured. A concentration of 0.05 M Tris was the safest for maintaining a pH between 8.0 and 8.2 (see Table 11).
4 . Reducing Agent Concentration Various concentrations of reducing agents were added to a constant volume of rehydrated FITC-labeled conjugates. The percentage remaining after 30 minutes of excitation (readings taken every minute) in the PerkinElmer spectrofluorophotometer is shown in Fig. 2. The error around each measurement was less than 1% and does not show up on the graphs. Data are shown for Dabco with rubella antibody and D l T with Toxoplasma antibody. D?T or DTE show the most protection, of the agents tested, when used at their optimal concentration of 0.033 M . Note that the optimal concentration for Dabco is 0.3 M, which is that recommended by Johnson et al. (1982).This is a concentration which is 10 times higher than that used for the other agents. Table 111 compares fading of the FITC-conjugate after 10 minutes of continuous excitation at the optimal concentration for each of the reducing agents Dabco and DTE with that of the unprotected conjugate. A 10-minute measuring period was chosen over the previously used 30-minute TABLE I1 OPrIMlZATlON OF
BUFFERCONCENTRATION
F O R DITIIIOTIIREITOI.
Tris concentration
0.20 M
0.10 M
0.05 M
0.02 M
8.38 8.38
8.24 8.25
8.14 8.21
8.03 8.11
15.93 18.77
10.83 14.19
10.72 12.57
10.66 10.20
PH With 0.2 M Dm Without D'IT Background intensitya With D'IT Without D?T a
Relative light intensity units.
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REDUCTION OF FADING
I
I
I
I
0
0.01
I
I
0.4
0.5
I
0.2 DABCO 0.3
0.1
I
0.03
I
I
I
0.02 D l T
0.05
0.04
Concentration (Mo!es/Liter)
FIG.2. Optimization of reducing agent concentration for protection from fading of FITClabeled antibodies after 30 minutes of excitation. The percentage remaining intensity in the Perkin-Elmer spectrofluorophotometer is plotted vs the reducing agent concentration in molestliter. Dabco was tested on rubella conjugate (open squares) and the DIT was tested on Toxoplusm gondii conjugate (open circles).
period in order to make the times better conform to those used in the microscope and those that might be used in a clinical laboratory.
5 . Microscope Verification of Protection After suitable protecting agents were found by screening in the macrofluorophotometer, the protective ability of these reagents was verified by TABLE I11
SELECTIONOF OPTIMAL REDUCING AGENTI N MACROFLUOROPHOTOMETEH ~ ~ _ _ _ _ _ _ _ _
DTE Background intensity" in 0.05 M Tris Optimum concentration in 0.05 M Tris
Conjugates Initial intensity minus background 10 minute intensity minus background Remaining intensity (%) based on initial intensity Remaining intensity (a) based on unprotected initial intensity
" Relative light intensity
units.
25.5 0.033 M
~~
Dabco
Unprotected
25.7 0.3 M
Not applicable
5.7
1049.3 918.4 87.8
701.5 654.5 94.0
1049.3 726.3 69.0
87.8
64.5
69.0
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G . L. PICCJOLO A N D D. S. KAPLAN
T i e (Min)
FIG. 3. Fading of cells on test kit slides with and without 0.025 M DTE added to buffered glycerol mounting medium in the Zonax. The percentage remaining intensity is plotted vs time in minutes for Toxoplasm (squares), rubella (triangles), and ANA (circles)with DTE (open) and without any reducing agent (solid).
incorporating the reducing agents into the mounting medium of the I F microscopy slides. Figure 3 is a plot of percentage remaining intensity after 10 minutes of continuous exposure to excitation light, with and without reducing agent present. Shown are results with T o x o p l a m , rubella virus, and ANA test kit slides mounted in kit buffered glycerol with or without 0.025 M DTE added. Since the labeled specimens fade too rapidly to record the initial, unfaded intensity, the highest intensity obtained with any of the protecting agents within the first measurement period (0.1 minute) (using the PMI program set to give 90 readings in a 10-minute maximum measuring period) was used as the initial intensity. Note that in the case of unprotected ANA and rubella virus only 8 and 5% (respectively) of the protected intensity remained after 0.1 minute. The initial intensity with DTE was 10 times greater than the unprotected intensity. Another interesting point is that when visually observing cells without DTE, they appeared totally red (due to counterstain) after 1 minute of continuous excitation. However, cells with DTE after 10
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REDUCTION O F FADING
Ot
I
l
l
l
1
2
3
4
l
I
5 6 Time IMin)
I
I
I
I
7
8
9
10
FIG.4. Effects of reducing agents on fading of FITC-labeled ANA cells with rhodamine counterstain during continuous excitation in the Zonax. The percentage remaining intensity vs the time in minutes of excitation is plotted. The ANA cells were mounted in buffered glycerol (triangles) and with 0.025 M DTE (squares)or 0.3 M Dabco (circles).
minutes of continuous excitation were still fluorescing bright green. Data similar to that shown in Fig. 3 were obtained using DT. But due to the inability of Tris buffer to maintain a pH of 8.2 in the DT solutions, this reducing agent was not used in future experiments. To verify the effectiveness of selected concentrations of reducing agents, cells with FITC-labeled conjugate to ANA with rhodamine counterstain were continuously excited in the Zonax. A comparison of percentage remaining intensity with 0.025 M DTE, 0.3 M Dabco, and no reducing agent in the buffered glycerol for 10 minutes of continuous excitation is shown in Fig. 4. Note that after 0.1 minute only 20% of the initial fluorescence intensity remained for Dabco. Even after 10 minutes of continuous excitation, 30%of the initial intensity remained for DTE as compared to 2.2 and 1.3% for Dabco and buffered glycerol, respectively. However, if Dabco is compared with its own intensity at 0.1 minute, it retains 11% of its own initial intensity which may indicate that fading is accelerated by Dabco initially but then the rate of fading slows. DTE was chosen for subsequent use on the basis of its
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G . L. PICCIOLO AND D. S. KAYLAN
80
60
-
OI
! CT
40
8
20
? 1
2
3
4
5
6
7
8
9
1
0
lime (Min)
FIG.5. Effects of DTE concentration on fading of FITC-labeled ANA cells with rhodarnine counterstain during continuous excitation in the Zonax. The percentage remaining intensity is plotted vs the time in minutes of excitation for cells with 0.025 M (squares), 0.033 M (circles), and 0.05 M (triangles) DTE added to the buffered glycerol.
higher initial fluorescence intensity and greater protective ability over the 10-minute measuring period, although DTT was almost as effective. Efforts were made to improve the protective ability of DTE without lowering the initial fluorescence intensity. Figure 5 shows the fading protection when 0.025,0.033, or 0.05 M DTE was incorporated into the mounting medium for the ANA IF microscopy kit. Note that there is almost a 40% lowering of the initial intensity with 0.05 M DTE. Also, 0.05 M DTE does not protect the sample better than 0.033 M DTE during the first 3.5 minutes of excitation. Therefore, 0.033 M DTE was chosen for use in further experiments due to the higher initial fluorescence intensity. The time required to align the specimen in the excitation field is normally less than 2 minutes during which 0.033 M DTE retains its protection.
6. Selection of Reducing Agent Based on the above-stated criteria, D l T and DTE were selected as the best protecting agents of those tested because they offered the most protec-
REDUCTION OF FADING
22s
tion from fading with the least inhibition of initial intensity. Note that 0.3 M Dabco lowered the initial intensity 23%.
D. DISCUSSION 1 . Optimization of Chemical Environment
Our data indicate that fading behavior of cell-free conjugates in the fluorometer is a good predictor of fading behavior of the fluorophore in the microscope-photometer.
a. Buffer Selection. Since the intensity of fluorescence is dependent on the pH of the medium (Nairn et aZ., 1969; McKay et aZ., 1981; Haiijman, 1977; Goldman, 1968; Jongsma et al., 1971), it is very important to choose a buffer that can maintain the optimal pH of 8.2-8.5. McKay, et al. (1981) found a 23% increase in the fluorescence of FITC upon raising the pH from 7.2 to 8.8. Jongsma et al. (1971) observed a 50% increase in the fluorescence intensity upon raising the pH from 6.0 to 7.0. These investigators also noted a 10%increase in fluorescence when the pH was raised from 7.0 to 8.0. After testing several buffers, including glycine, Tris, and carbonate, Tris buffer proved to maintain the pH in the presence of reducing agent and also to have the lowest background intensity.
b. Reducing Agent Selection. The ultimate usefulness of reducing agents is to inhibit fading while the specimen is being examined microscopically. Therefore, those reducing agents employed must be able to function with the buffer and pH of the mounting medium. DTE was selected as the best reducing agent based upon the above-stated selection criteria. DTE significantly reduced fading over a 10-minute continuous excitation period and did not reduce the initial intensity of the fluorophore. In addition, DTE is easy and practical to use with the buffered glycerol mounting medium. Based on the results shown in Fig. 2 with Dabco, it is possible that the use of a lower concentration than that tested by Johnson (i.e., 5 0.1 M ) would result in only a 10% increase in fading, while the initial intensity would be much higher since high concentrations of Dabco suppress the fluorescence intensity. c. Reducing Agent Concentration. It was necessary to determine the lowest concentration of reducing agent that could prevent fading for several reasons. First, if the concentration of the agent were too high, reduction of the initial fluorescence may occur, possibly causing faulty end-point determination. Second, the concentration must be low so that background inten-
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G. L. PICCIOLO A N D D. S . KAPLAN
TABLE IV MOUNTINGMEDIUMFOR O P ~ M APROTECTION L Find concentration in buffered glycerol
Tris concentration DTE concentration
0.05 M
PH
8.0-8.2
0.033 M
sity is not increased due to autofluorescence of the reducing agent or precipitation of salts on the slide. Table IV gives the final reducing agent concentration and pH that we recommend for incorporating DTE into buffered glycerol. Additional stability studies are underway to determine conditions of use for DTE, which is labile in solution at room temperature. 2. Kinetics
Comparing the slopes of the linear regression lines for samples with different reducing agents allows a quick and precise method of comparing the rate of decrease in intensity over a given time period (Table V). Dabco shows a more negative value than the other reducing agents tested which indicates greater fading. DT, D'IT, and DTE show very similar slopes and thus are equivalent in protective function when used at their own optimal concentration. Figure 6 shows the change in fluorescence intensity as a function of excitation time of the labeled rubella infected cells (from the test kit) mounted in buffered glycerol or with DTE added. When DTT or DTE is present, regression analysis indicated that the best fit is a straight line, while with no protection, the best fit is a quadratic curve. This implies that with the reducing agent present, the reaction is first order (or a function of one ratelimiting factor) and that without it, the reaction is due to the interaction of two rate-limiting factors. This difference may represent two different mechaTABLE V WITH
Agent Dahco DT DlT DTE
SLOPE OF FADING DIFFERENT REDUCINGAGENTS Conc. (M)
(% per second)
0.3 0.025
-17.02 -4.64
0.025
-4.30 -4.18
0.ow
Slope
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REDUCTION OF FADING
tI 0
I
1
I
I
I
I
I
I
I
1 2 3 4 5 8 1 8 9 1 0 Time (Min)
FIG.6. Best fit regression curves for fading of FITC-labeled rubella infected cells during continuous excitation when mounted in buffered glycerol (open circles) or with 0.05 M DTE (solid circles) added.
nisms of fading, an oxygen-sensitive mechanism and a non-oxygen-sensitive mechanism. We hypothesize that fading in the presence of a reducing agent is not oxygen sensitive. But it should be pointed out that the reducing agents only scavenge the oxygen and that in no way in our experiments have exhaustive measures been taken to completely remove oxygen from the mounting medium. Our results over the past 2 years did not always show a linear response in the presence of a reducing agent. Further work to clarify the inconsistency is underway. One possibility is that since the amount of water in the glycerol from various kits varies (from 10 to 75%) the concentration of dissolved oxygen also varies and it interacts with the concentration of reducing agent. Another possibility is that in the presence of a hydrogen donor, the reducing agent, photoreduction of the excited fluorophore occurs. The slower rate of fading in the presence of the reducing agents is, then, a result of the photoreduction of the flurorophore.
3. Use with Other Systems Verification of the effectiveness of DIT for protection from fading was performed using mouse myeloma, Syrian hamster embryo, and BT16 transformed cells by S . Grillo of Johns Hopkins University. With fluoresceinamine, which adsorbs to cell membranes, the effects of continuous exci-
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G . L. PICCIOLO AND D . S. KAPLAN
tation in the Zonax were determined. Within the first minute, 90-95% of the fluorescence was lost without protection, while in samples protected with 0.033 M DTT in 0.05 M Tris, only 5-10% was lost after 16.2 minutes. Higher initial intensities for the protected cells were also observed, which allow an increased fluorescence signal for better reproducibility and use in day-to-day comparisons. This is important when attempting in situ hybridization studies which provide a small signal (S. Grillo, personal communication).
4 . Mechanism Speculations The exact mechanism of fluorescence fading as a function of excitation energy and irradiation time is not known. We postulate (Fig. 7) that DTE reduces fading by scavenging up the free oxygen in the mounting medium and preventing the reaction of the triplet state of fluorescein with molecular oxygen, which would result in an oxygenated, nonfluorescent species of
Intersystem Croas
Ground State
Oxidized Fluwophore
FIG.7. Proposed fluorescence conversions showing relative energy levels. Excitation light excites ground state fluorophore (FL-So) to excited singlet (FL-Sl). Light emission occurs with decay to ground state or intersystem crossing to triplet (FL-TI)and then decay to ground state. Radiationless decay by interaction with oxygen to oxidized fluorophore (FL-0) is inhibited in the presence of reducing agents.
REDUCTION OF FADING
229
fluorescein. The excited fluorescein molecules are now able to return to the singlet, ground state, through normal decay mechanisms, with the production of light and are available for a repeat of the excitation cycle. This hypothesis agrees with Giloh and Sedat (1982) who postulated that scavenging agents such as PPD, ascorbic acid, and nPG prevent fading by removing free oxygen from the mounting medium. Vaughan and Weber (1970) also showed that molecular oxygen decreases the fluorescence lifetime of solutions. In accordance with Fukuda et al. (1980), it is possible that by removing the oxygen from the mounting medium, the pH of the medium is stabilized which results in strengthened bonding between the FITC and the immunoglobulin. This results in less availability of the fluorophore to the photodecomposition process and in decreased fading of the FITC molecules. The curve shapes of some of the fading results reported by Johnson et al. (1982) are the same as in Fig. 6, i.e., linear with the reducing agent and a quadratic curve without the reducing agent. They, however, performed additional experiments to demonstrate the role of oxygen in fading from which they conclude that singlet oxygen is not involved in the fading mechanism. One of the technical problems is the difficulty in removing all traces of oxygen in an aqueous environment. Whether this accounts for the differences must be elucidated by exhaustive oxygen removal via other techniques ( D . Benson, personal communication). Further research is necessary to elucidate the mechanism of protection of various agents.
VI. Summary A. PROGRESS IN REDUCING FADING OF FRP Many researchers have used a variety of techniques to reduce fading of the specimen during excitation. Table I shows the techniques used and the principal investigators. Each of these techniques has advantages and disadvantages. In most instances it is not possible to directly compare fading rates obtained by the various methods since fading varies directly with the intensity of the exciting light. With the exception of the use of lasers, most investigators do not have any means of measuring the intensity of the exciting light and, therefore, they do not report a power density flux. 1. Localization under Phase-Contrast
Many specimens can be located under phase contrast or other nonfluorescence exciting illumination, but some cannot (e.g., the treponemes). In addition, one cannot tell if the specimen to be measured is positive or negative or if there is fluorescent debris near the specimen. Searching to be sure that the specimen does not contain any positive cells requires fluorescence excitation.
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G. L. PICCIOLO A N D D. S. KAPLAN
2. Epijluorescence Shutter The use of an epifluorescence shutter allows the specimen to be excited for milliseconds (Geyer et al., 1978). This allows repeated measurements on the same sample with relatively little fading (Kaufmann et d.,1971; Nairn et aZ., 1969; McKay et al., 1981; Jongsma et al., 1971).
3. Pre- or Postirradiation Fukuda pre- or postirradiated the Feulgen-stained nuclei to eliminate primary (nonspecific) fluorescence. This method is not practical for use by the clinical laboratory when dealing with clinical specimens since it is timeconsuming. Also, FITC fades very rapidly and postirradiation of the specimen would reduce the fluorescence signal which could result in false-negative readings. 4. Field Diaphragm
These diaphragms limit the amount of exciting light that illuminates the specimen. The advantage of using field diaphragms is that they reduce the fluorescence of neighboring cells while measuring a specimen. Therefore, the total field is not faded by measurement and the neighboring cells are available for quantitation. They also reduce the background fluorescence since one can adjust the excited field area to be just slightly larger than the specimen (Kaplan and Picciolo, 1984).
5. Chemical Agents Agents such as DTE, nPG, PPD, or Dabco can be incorporated directly into the mounting medium for the I F microscopy slides. These agents retard fading of the FITC-labeled conjugate and, especially when used in combination with the epifluorescence shutter, allow reproducible measurements of the same sample for several minutes. We have optimized the use of a chemical reducing agent, DTE, by incorporating it into the mounting medium at a final concentration of 0.033 M in 0.05 M Tris (pH 8.2). This allows for results of 75% remaining intensity after 1 minute which can be of practical use in the clinical or research laboratory (Kaplan et al., 1981; Kaplan and Picciolo, 1982, 1983; Picciolo and Kaplan, 1982, 1983).
B. MECHANISMOF PROTECTION Any factors that increase the exposure of FRP to excitation energy increase its fading. However, by reducing opportunities for the reaction of FRP with oxygen (by reducing the oxygen concentration, removing water with its dissolved oxygen, the addition of sulfhydryl-containing agents to
REDUCTION OF FADING
23 1
scavenge oxygen, increasing the conformational stability of FRP so as to reduce its collisions with oxygen), one can reduce fading during excitation. The possibility that oxygen is not involved in the fading reaction remains to be definitively demonstrated, although Johnson et al. (1983) have reported this interpretation of their data.
C. IMPROVEMENTS FOR FUTURE USE Reduction of fading would significantly improve quantification of FRP on IF microscopy slides. Retarding fading would permit longer scan times on slides without concomitant decreases in fluorescence intensity. Therefore, weakly positive cells could be scanned without the fear of these cells losing their fluorescence and being interpreted as negative. This increased ability to scan without fading would permit the introduction of automated or semiautomated instrumentation which could scan a slide and determine the end point quantitatively. By the use of such methods to retard fading of the fluorescent reaction product, the use of scanning methods of image analysis and quantification of the intensity of the FRP would accurately reflect the starting intensity and thus enable many more applications of this technology. Advances in utilizing IF technology in many areas of application were recently presented at the Fourth International Conference on Automation of Diagnostic Cytology, Montreal, Province of Quebec, Canada, June 24-25, 1983. Subsequent articles and abstracts were published in Analytical and Quantitative Cytology Vol. 5(3), 1983. REFERENCES BelIin, J. S. (1968). Photophysical and photochemical effects of dye binding. Photochem. Photobiol. 8 , 383-392. Bergquist, N. R. (1973). The pulsed dye laser as a light source for fluorescent antibody technique. Scand. ]. Immunol. 2, 37-44. Bergquist, N. R . , and Nilsson, P. (1975). Laser excitation of fluorescent copolymerized immunoglobulin beads. Ann. N. Y. Acad. Sci. 254, 157-162. Bohm, N., and Sprenger, E. (1968). Fluorescence cytophotometry: A valuable method for quantitative determination of nuclear Feulgen-DNA. Histochemie 16, 100-118. Enerback, L., and Johansson, K. (1973). Fluorescence fading in quantitative fluorescence microscopy: A cytofluorometer for the automatic recording of fluorescence peaks of very short duration. Histochem. J . 5, 351-362. Fourth International Conference on Automation of Diagnostic Cytology, June 24-25, 1983, Montreal, Canada. Anal. Quant. Cytol. Vol. 5(3) (1983). Fujita, S. (1973). DNA cytofluorometry on large and small cell nuclei stained with pararosaniline Feulgen. Histochemie 36, 193-199. Fujita, S . , and Fukuda, M. (1974). Irradiation of specimen by excitation light before and after
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staining with pararosaniline Feulgen: A new method to reduce non-specific fluorescence in cytofluorometry. Histochemistry 40, 59-67. Fukuda, M., Isemura, T., Maruo, N., Nakanishi, K., and Fujita, S. (1975). Cytofluorometric measurement of contents of nuclear DNA and intracellular prophyrin converted from heme or hemoglobin on a single erythroid cell. Acta Histochem. Cytochem. 8, 331-341. Fukuda, M., Nakanishi, K., Mukainaka, T., Shima, A., and Fujita, S. (1976). Combination of Feulgen nuclear reaction with immunofluorescent staining for photoproducts of DNA aRer UV-irradiation. Actu Histochem. Cytnchem. 9, 180-192. Fukuda, M., Nakanishi, K., Sawamura, I., and Fujita, S. (1977). Standardization of postirradiation method to eliminate primary fluorescence in cytofluorometry. Histochemistry 52, 119-127. Fukuda, M., Tsuchihashi, Y., Takamatsu, T., Nakanishi, K., and Fujita, S. (1980). Fluorescence fading and stabilization in cytofluorometry. Histochemistry 65, 269-276. Geyer, M. A., Dawsey, W. J., and Mandell, A. J. (1978). Fading: A new cytofluorimetric measure quantifying serotonin in the presence of catecholamines at the cellular level in brain. 1.Pharmacol. E x p . Ther. 207, 650-667. Gill, D. (1979). Inhibition of fading in fluorescence microscopy of fixed cells. Experientia 35, 4OO-40 1. Giloh, H., and Sedat, J. W. (1982). Fluorescence microscopy: Reduced photobleaching of rhodamine and fluorescein protein conjugates by n-propyl gallate. Science 217, 1252-1255. Golden, J. F., and West, S . S. (1974). Fluorescence spectroscopic and fading behavior of Ehrlichs hyperdiploid mouse ascites tumor cells supravitally stained with acridine orange. J. Histochern. Cytochem. 22, 495-505. Goldman, M. (1968). “Fluorescent Antibody Methods.” Academic Press, New York. Guilbault, G. G. (ed.) (1967). “Fluorescence: Theory, Instrumentation and Practice.” Dekker, New York. Haaijman, J. J. (1977). “Quantitative Immunofluorescence Microscopy: Methods and Applications.” Inst. Experim. Gerontol. TNO, Rijswijk (Z.H.), The Netherlands. Herzog, F., Albini, B., and Wick, G. (1973). Comparison of filters used in irnmunofluorescent staining procedures with fluorescein-isothiocyanate (FITC) conjugates. J . Zmmunol. Methods 3, 211-220. Johnson, G. D., and de C. Nogueira Araujo, G. M. (1981). A simple method of reducing the fading of immunofluorescence during microscopy. J . Zrnmunol. Methods 43, 349-350. Johnson, G. D., Davidson, R. S., McNamee, K. C . , Russell, G., Goodwin, D., and Holborow, E. J. (1983). Fading of immunofluorescence during microscopy: A study of the phenomenon and its remedy. J . Zmmunol. Methods 55, 231-242. Jongsma, A,, Hijmans, W., and Ploem, J. S. (1971). Quantitative immunofluorescence: Standardization and calibration in microfluorometry. Histochemie 25, 329-343. Kaplan, D. S., and Picciolo, G. L. (1982). Standardization of imrnunofluorescent tests by quantitative microfluorometry: Reduction in fading of fluorescein isothiocyanate labeled antibody using chemical reducing agents. American Society for Microbiology Meeting Ahstract. Kaplan, D. S., and Picciolo, G. L. (1983). Use of computer controlled microscope photometer to quantitatively analyze intensity images of immunofluorescent cells. American Society for Microbiology Meeting Abstract. Kaplan, D. S., and Picciolo, G. L. (1984). Characterization of instrumentation and calibrators for quantitative microfluorometry for immunofluorescence tests. In preparation. Kaplan, D. S., Picciolo, G. L., and Stauffenberg, R. (1981). Standardization of immunofluorescent tests by quantitative microfluorometry: Use of fluorescent particles for instrument calibration. American Society for Microbiology Meeting Abstract.
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Kaufman, G. I., Nester, J. F., and Wasserman, D. E. (1971). An experimental study oflasers as excitation sources for automated fluorescent antibody instrumentation. J . Histochem. Cytochem. 19, 469. Lea, D. J . , and Ward, D. J. (1979). Control of excitation in the fluorescence microscope. J . Zmmunol. Methods 31, 191-192. McCarthy, W. J . , and Moyer, E. S. (1970). Fundamental concepts of fluorescence and phosphorescence. In “Introduction to Quantitative Chemistry-11” (G. L. Weid and G . F. Bahr, eds.), pp. 399-429. Academic Press, New York. McKay, I. C., Forman, D., and White, R. G. (1981).A comparison offluorescein isothiocyanate and lissamine rhodamine (RB 200) as labels for antibody in the fluorescent antibody technique. Immunology 43, 591-602. Menter, J. M., Golden, J. F., and West, S. S. (1978).Kinetics offluorescence fadingofacridine orange-heparin complexes in solution. Photochem. Photobiol. 27, 629-633. Menter, J. M., Hurst, R. E., and West, S. S. (1979). Photochemistry of heparin-acridine orange complexes in solution: Photochemical changes occurring in the dye and polymer on fluorescence fading. Photochem. Photobiol. 29, 473-478. Nairn, R. C., Herzog, F., Ward, H. A., and De Boer, W. G. R. M. (1969). Microphotometry in immunofluorescence. Clin. Exp. Zmmunol. 4, 697-705. Pearse, A. G . E., and Rost, F. W. D. (1969). A microspectrofluorimeter with epi-illumination and photon counting. 1. Microsc. (Oxford) 89, 321-328. Picciolo, G. L., and Kaplan, D. S. (1982). Immunofluorescence standardization by quantitative microfluorometry: 1. Standards for calibration. 11. Reduction in fading by reducing agents. Immunol. Commun. 12, 106. Picciolo, G. L., and Kaplan, D. S. (1983). Computer-controlled quantitative microfluorometry of serologic immunofluorescence. Anal. Quant. Cytot. 5, 214. Piller, H. (1977). “Microscope Photometry.” Springer-Verlag. Berlin and New York. Ploem, J. S. (1967). The use of a vertical illuminator with interchangeable dichroic mirrors for fluorescence microscopy with incident light. Z. Wiss. Mikrosk. 68, 129-142. Ploem, J. S. (1970). Quantitative immunofluorescence. In “Standardization in Immunofluorescence” (E. J. Holborow, ed.), pp. 63-73. Blackwell, Oxford. Ploem, J. S. (1971). A study of filters and light sources in immunofluorescence microscopy. Ann. N . Y. Acad. Sci. 177, 414-429. Ploem, J. S. (1975). Introduction. Ann. A’. Y. Acad. Sci. 254, 4-20. Ploem, J. S. (1982). Automated methods in immunofluorescence studies. I n “Immunofluorescence Technology: Selected Theoretical and Clinic4 Aspects” (G. Wick, K. N. Trail, and K. Schauenstein, eds.), pp. 73-94. Elsevier, Amsterdam. Ploem, J. S., de Sterke, J. A., Bonnet, J., and Wasmund, H . (1974).A microspectrofluorometer with epi-illumination operated under computer control. J. Histochem. Cytochem. 22,668677. Rigler, R.,Jr. (1966). Microfluorometric characterizatrion of intracellular nucleic acids and nucleoproteins by acridine orange. Acta Physiol. Scand. 67 (Suppl. 267), 117. Rodriguez, J . , and Deinhardt, F. (1960). Preparation of a semipermanent mounting medium for fluorescent antibody studies. Virology 12, 316-317. Schauenstein, K., Wick, G . , Herzog, F., and Steinbatz, A. (1975). Investigation of the recovery phemenon in immunofluorescence after laser excitation. J. Immunol. Methods 8, 9-16. Schauenstein, K., Bock, G., and Wick, G. (1978).Factors influencing fluorescence properties of free and protein-bound fluorochromes. Macro- and microfluorometric observations. In “Immunofluorescence and Related Staining Techniques” (W. K. Knapp, Holubar, K., and G. Wick, eds.), pp. 81-95. Elsevier, Amsterdam. Schauenstein, K., Bock, G., and Wick, C. (1980). Short time bleaching of fluorescein isothio-
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cyanate: A possible parameter for the specific binding of conjugates in immunofluorescence. J. Histochern. Cytochem. 28, 1029-1031. Schauenstein, K., Bock, G., and Wick, G. (1982). The use of lasers to determine the fluorescence characteristics of fluorescein derivatives in immunofluorescence assays. In “Imrnunofluorescence Technology: Selected Theoretical and Clinical Aspects” (C. Wick, K. N. Trail, and K. Schauenstein, eds.), pp. 27-36. Elsevier, Amsterdam. Sernetz, M., and Thaer, A. (1973). Microcapillary fluorometry and standardization for microscope fluorometry. In “Fluorescence Techniques in Cell Biology,” pp. 41-49. SpringerVerlag, Berlin and New York. Taylor, C. E. D., and Heimer, G. C. (1974). Measuring immunofluorescence emission in terms of standard international physical units. J . B i d . Stand. 2, 11-20. Thaer, A. A. (1966). Instrumentation for micrafluorometry. In “Introduction to Quantitative Cytochemistry” (G. L. Weid, ed.), Vol. 1, pp. 409-426. Academic Press, New York. Udenfriend, S. (ed.) (1962). “Fluorescence Assay in Biology and Medicine.” Academic Press, New York. Vaughan, W. M., and Weber, G. (1970). Oxygen quenching of pyrenebutyric acid fluorescence in water. A dynamic probe of the microenvironment. Biochemistry 9, 464. Wick, G . , Schauenstein, K., Herzog, F., and Steinbatz, A. (1975). Investigations of the recovery phenomenon after laser excitation in irnmunofluorescence. Ann. N. Y. Acad. Sci. 254, 172-174. Zeiss, Inc. (1983). “Worthwhile Facts about Fluorescence Microscopy.” Carl Zeiss 7082 Oberkochen, West Germany.
INDEX
A Absidia glauca, PAH metabolism, 36 pseudocylindrospora, PAH metabolism, 36 ramosa, PAH metabolism, 36 spinosa, PAH metabolism, 36 Acanthamoeba, in drinking water, 107, 108 Achromobactcr metabolism of PAH, 35 in potable water, 91 in water supply systems, 93 Acinetobacter calcoaceticus, in water supply systems, 93 in drinking water, public health importance, 98-99 in water supply systems, 93, 96 Acremonium, from drinking water, 102 Acridine orange, to elucidate mechanism of fading, 213 Actinomycetes in drinking water, 100 importance of, 101 on drinking water distribution system wall/pipe surfaces, 101 effect of water treatment practices on, 86 in water supply systems, 93 Adenovirus, 159 removal, during sludge treatment, 157 Aeroinonas hydrophila in drinking water, public health importance, 98-99 in water supply systems, 93 metabolism of PAH, 35 naphthalene metabolism, 43-44 in water supply systems, 93, 96
235
Aginenellum quadruplicatum naphthalene metabolism, 45-46 PAH metabolism, 37, 38 Alcaligenes in potable water, 91 in water supply systems, 93, 96 Algae in drinking water, 105-106 importance, 106-107 on drinking water distribution system wall/pipe surfaces, 106 in drinking water treatment plants, 87 naphthalene metabolism, 45-46 PAH metabolism, 37, 40-41, 64 Alternaria from drinking water, 102 on pipe surfaces, 103 ldminopyrene, microbial metabolism, 62 Amoebae, in drinking water, 107. See also Entamoeba Amphora, PAH metabolism, 37 Anabaena, PAH metabolism, 37 Ankistrodesmus, 106 Anthracene bacterial oxidation, pathway, 47 biodegradation, in natural habitats, 63 fungal oxidation, pathway, 48-49 microbial metabolism, 38, 46-51 structure, 32 Aphanocupsa, PAH metabolism, 37 Arthrobacter, in water supply systems, 93 Aspergillus niger, PAH metabolism, 36 ochraceus benzo[a]pyrene hydroxylase, 53, 55 PAH metabolism, 36
236
INDEX
B Bacillus, in water supply systems, 93 Bacteria chlorine tolerance, 111-112 on distribution system wall/pipe surfaces, 91 in drinking water, 90-96 public health importance, 98-100 on drinking water distribution system wall/pipe surfaces, 96-98 oxidation of 3-methylcholanthrene, pathways, 60-61 oxidation of aromatic hydrocarbons, 34-40 PAH metabolism, 64 removal, from drinking water, 82-86 in treated water, as water moves from source to consumer, 92-96 types, in water supply systems, 92-94 Bacteriophage adsorption, 6-7, 138, 148-149 adsorptive behavior, 142-143 concentration, after wastewater chlorination, 151-152 control, 16-25 in fermentation, 2-3 destructive role in milk fermentations, 1-2
f2 adsorptive behavior to soil, 143 disinfection, 152 inactivated in fluids, 154 removal, from wastewater, 157 host-controlled modification, 6-7 host interactions, 4-16 host range, 5-6 inhibitory media, 16-17 interactions with lactic streptococci, 1-29 isometric, 4 lytic development, 4-13 morphology, 4 MS-2 adsorption, to bentonite, 137 adsorptive behavior in soil, 143 coat protein sequence, 143 degradation, 154 disinfection, 152 inactivated in fluids, 154 isoelectric points, 142 multiplication, and beta-glycerophosphate in growth medium, 5, 9
mutation, 6 origin, during milk fermentations, 16 phage assays, buffered media, 5 prolate, 4 4X-174, adsorptive behavior in soil, 137, 143 R17, adsorption, to allophane, 137 removal, from water, 158 replication, 8-10 calcium-dependent, 8 host-dependent, 10-13 requirement for electrolytes, 8 temperature conditions, 9-10 T1 adsorption, 146, 149 inactivation, 151 T2 adsorption, and pH, 147 adsorptive behavior to soil, 143 isoelectric points, 142 T4, 160 adsorption, 160 adsorptive behavior to soil, 143 isoelectric points, 142
T7 adsorption, 146, 149 inactivation, 151, I52 Balantidizrm coli, in drinking water, 108 Basidioboh ranarum, PAH metabolism, 36 Beuerinckia anthrdcene metabolism, 48 benz[a]anthracene metabolism, 55-56 metabolic activation of 3-methylcholanth ren e , 60 oxidation of benzo[a]pyrene, 52-53 oxidation of phenanthrene, 50 PAH metabolism, 35, 38 Benz[a]anthracene, 46, 52 alkyl-substituted, microbial metabolism, 57-61 biodegradation, in natural habitats, 63 carcinogenicity, 55 trans-3,4-dihydrodiol, 55-56 trans-3,4-dihydrodiol-I,2-epoxide, 55-56 fungal oxidation, pathways, 56-57 mammalian metabolism, 55 microbial metabolism, 38, 55-57 mutagenicity, 55 structure, 32 tumorigenicity, 55
237
INDEX
Benzene, microbial metabolism, 35-40 Benzene dioxygenase, 41 Benzo[a]pyrene, 46 biodegradation, in natural habitats, 63 mammalian metabolism, 51-52 microbial metabolism, 35-40, 51-55 structure, 32 BIONET, 194 Bloodworms, in drinking water distribution systems, 108 Bodo, on reservoir surfaces, 108
C
Campylobacter jejuni, disinfection, from drinking water, 85 Candida albicans, as indicator organism in water quality analysis, 105 guilliennondii, PAH metabolism, 36 lipolytica, PAH metabolism, 36 maltosa, PAH metabolism, 36 tropicalis, PAH metabolism, 36 Cephalosporium from drinking water, 102 on pipe surfaces, 103 Chemisorption, 134 Chlamydomonas angulosa, PAH metabolism, 37 Chlorella, 106 autotrophica, PAH metabolism, 37 sorokiniana, PAH metabolism, 37 Chlorine. See also Water, chlorination algicidal, 107 for fungal control, 103 Choanephora campincta, PAH metabolism, 36 Chrombacteriurn, in water supply system, 93 Circinelka, PAH metabolism, 36 Citrobacter in drinking water, public health importance, 98-99 in water supply systems, 93 Cladosporium, on pipe surfaces, 103 Clams, in drinking water distribution systems, 108 Claoiceps paspali, PAH metabolism, 36 Coccochloris elabens, PAH metabolism, 37
Cokeromyces poitrussi, PAH metabolism, 36 Coliforms in drinking water, 91, 99 heterotrophic plate count, 99 as indicator of source water microbial quality, 77-78 research needs, 114 removal, from drinking water, 83-85 and risk of illness of viruses and Salmonella, 115-116 standard plate count, 99 on surfaces of drinking water distribution system, 97-98 in water supply systems, 96 Colloid stability, DLVO theory, 135 Computers display of genetic information, 183-184 expert systems, 191-193, 194 in genetic engineering, 169-195 as tool for scientist, 169 Computing environment, 170-172 for genetic engineering, 172-185 Conidiobolus gonimodes, PAH metabolism, 36 Corynebacterium renale metabolism of PAH, 35 naphthalene oxygenase, 43 Coryneform, in water supply systems, 93 Coxsackie virus A2, removal, from water, 158 A3, removal, from water, 158 A9, removal, from wastewater, 156-157 A21, isoelectric points, 142 B1, 157 B3 adsorption, to activated sludge floes, 157 adsorptive behavior to soil, 143 concentration, 162 B4 adsorptive behavior to soil, 143 concentration, 162 B5, 157 cyptococcus albidus, on water pipes, 105 laurentii, from drinking water, 104-105 Cunninghamella bainieri benzo [alpyrene hydroxylase, 53-55 PAH metabolism, 36, 38
238
INDEX
blakesleeana, PAH metabolism, 36 echinulata, PAH metabolism, 36 elegans, 56 anthracene metabolism, 48 benzo[a]pyrene hydroxylase, 53 formation of proximate carcinogen of benz[a]anthracene, 57 metabolic activation of 3-methylcholanthrene, 60 metabolism of benzo[a]pyrene, 53-54 methylbenz[a]anthracene metabolism, 58 4-methylbenz[a]anthracene metabolism, 59 naphthalene metabolism, 45 PAH metabolism, 36, 38 phenanthrene metabolism, 50-51 UDP-glucrironosyltransferase, 45 japonica, PAH metabolism, 36 Curnularia lunata, PAH metabolism, 36 Cyanobacteria, PAH metabolism, 37, 40, 45-46, 64 Cyclops, in drinking water distribution systems, 108 Cylindrotheca, PAH metabolism, 37 Cytophaga, in water supply systems, 93
D Debaryornyces hansenii, PAH metabolism, 36 Deoxyribonucleic acid, complementation (cDNA), computer assisted analysis, 182-183, 187-191 Diatoms. See also spec+ species PAH metabolism, 64 1,4-Diazabicyclo[2.2.2]octalie, to reduce fading, comparison studies, 217-230 I)il)enz[a,h]anthracene, biodegradation, i n natural habitats, 63 cis-Dihydrodiols, microbial metd>olism, 3540 7,12-Di1nethylbenz[a]anthracene, niicrohial metabolism, 57-61 12-Dirnetliylhenz[n]anthr~cene, structure, 32 I)ithioerythritol, to reduce fiading, comparison studies, 217-230
1)ithiothreitol optimization of buffer concentration, 220 to reduce fading, comparison studie5, 217-230 DNACE, 170-194 applications addressed in, 172-173 DNAHELP library, 174-175 information storage and management, 176-178 routine computation, 177-179 sequence manipulations and analysis, 177-183 Double-layer theory, 134-135 Dunaliella tertiolectra, PAH metabolism, 37
E Echinamoeba, in drinking water, 107 Echovirus 1 adsorption, to estuarine sediments, 137 adsorptive behavior in soil, 143 concentration, 162 inactivation, in sediments, 151 isoelectric points, 142 Echovirus 7, 157 adsorption, to activated sludge floes, 157 adsorptive behavior to soil, 143 Echovirus 29, adsorption, to activated sludge floes, 157 Ecosystem, definition. 75 Edwarhiella tarda, in drinking water, public health importance, 98-99 Emericellopsis, PAH metabolism, 36 Entamoeba histolytica, in drinking water, 108 in water, 87-88 Enteric virus concentration, after wastewater chlorination, 151-152 detection, in activated sludge, 156 Enterobacter aerogenes, in water supply systems, 93 agglomerans, in water supply systems, 93 cloacae in source water, 78 in water supply systems, 93 in drinking water, public health importance, 98-99 in water supply systems, 96
INDEX
239
Enteroviruses adsorptive behavior, 142-143 in groundwater, 161 inactivation, protection against, 152 removal during sludge treatment, 157 from water, 158 survival, 149 Epicoccum from drinking water, 102 nigrum, PAH metabolism, 36 on pipe surfaces, 103 Escherichia coli, 44 removal, from drinking water, 83-85 in drinking water, public health importance, 99 resistance to ozone, 103 survival, in water, 78 Euplotes, on reservoir surfaces, 108 European Molecular Biology Laboratory’s Nucleotide Sequence Data Library, 176
F Filamentous fungi chlorine resistance, 86 in drinking water, 101-104 importance, 103-104 on drinking water distribution system wall/pipe surfaces, 102-103 effect of water treatment practices on, 86 resistance to ozone, 103 Flavobacterium chlorine tolerance, 112 in drinking water, 91 public health importance, 98-99 PAH metabolism, 35, 38 in water supply systems, 93, 96 Fluorescein isothiocyanate (FITC) conjugates, 208 fading measurements, 205-208 mechanism, 214-215 for fading measurement, 218 Fluorescein isothiocyanate (F1TC)-labeled antibody, 198 fading, 214-215
Fluorescence fading, 197-198 advantages of reducing, 199 argon ion laser measurements, 205 and chemical agents, 230 and collector lens, 202 comparison, 205-215 comparison of protecting agents, 215229 and conventional light sources, 206208 definition, 198 environmental factors affecting, 204 and epifluorescence shutter, 230 and excitation and neutral density filters, 203 and excitation energy, 202 and excitation source, 205-208 and excitation time, 204 and field diaphragms, 230 and heat filters, 203 kinetics, 226-227 and lamp housing, 201 laser experiments, 205-206 and light source, 201-202 measurement, 218-219 mechanism, 211-215 speculations on, 228-229 mechanism of protection, 230-231 and mercury lamps, 206-207 methods for reducing, 198 and objectives, 203-204 and pre- or postirradiation, 230 progress in reducing, 229-230 protection, microscope verification of, 221-224 pulsed dye laser comparisons, 205-206 reducing agent concentration, 220-221, 225-226 selection of reducing agent, 224-225 and specimen localization under phasecontrast, 229 statistical analyses, 219 and xenon lamps, 207-208 intensity and buffer selection, 225 factors affecting, 201-204 measurements, instrumentation, 19920 1 optical factors, 201-204
240
INDEX
methods of protection, 208-211 by pre- or postfixation of specimen, 210-211, 212-213 with chemical agents, 208 with fixation in a nonfluorescent resin, 210 optimization of chemical environment, 225-226 principle, 211-212 recovery, 206, 208, 212 stabilization with 1,4-diazabicyclo[2,2.2]octane, 209 with p-phenylenediamine, 209 with n-propyl gallate, 208-209 with sodium dithionite, 208 Fungi. See also Filamentous fungi anthracene metabolism, 48 benz[a]anthracene metabolism, 56-57 benzo[a]pyrene metabolism, 52-53 naphthalene metabolism, 44-45 oxidation of 3-methylcholanthrene, pathways, 60-61 oxidation of aromatic hydrocarbons, 36, 40 PAH metabolism, 64 phenanthrene metabolism, 50-51 Fusarium from drinking water, 102 on pipe surfaces, 103
Gliocladium, PAH metabolism, 36 Gouy layer, 134-135 Groundwaters, disinfection, 79
H Hartmunnello, in drinking water, 107 Helicostylum pirifonne, PAH metabolism, 36 Hepatitis A virus, 133 Herpes virus hominis type 1, adsorption, 143, 148 Hyphochytrium catenaides, PAH metabolism, 36
I Influenza A, isoelectric points, 142 Insects, in drinking water distribution systems, 108 INSIGHT gene, 185-193 Ions antichaotropic, 138-139 chaotropic, 138-139
K G GenBank Genetic Sequence Data Bank, 176-177 Genetic engineering analysis of expression, computers in, 180182 automation, 194 computational support systems, 170-194 computer support, 169-170 expert systems, 192-193 model project, computer applications in, 185-193 Giardia lumblia, in drinking water, 108 survival, in water, 78 in water, 87-88 Gilbertella persicaria, PAH metabolism, 36 Glenodinium, 106
Klebsiellu in drinking water, public health importance, 98-99 pneumoniae, in drinking water, public health importance, 98-99 in water supply, 79, 93, 96
L Lactobacillus casei S - 1 , temperate and lytic bacteriophages, 13-14 in water supply systems, 94 Langmuir isotherm, 136-137 Legionella in distribution systems, 116 in drinking water, public health importance, 98-99
241
INDEX
pneumophila chlorine tolerance, 112 disinfection, from drinking water, 85 survival, in water, 78 kptothrix, in water supply systems, 93 Lilly Company DNA Computing Environment. See DNACE Lilly Interactive Drug Design System, 172 Lilly Research Laboratories Research Computing Environment, 172
M Macrofluorophotometer, 199-200 measurement of fading in, 218 selection of optimal reducing agent in, 220-221 4-Methylbenz[a]anthracene fungal oxidation, pathways, 59-60 microbial metabolism, 38 7-Methylbenz[a]anthracene fungal metabolism, 58 mammalian metabolism, 58 microbial metabolism, 38, 57-61 structure, 32 Methylbenz[a]anthracenes, microbial metabolism, 57-61 3-Methylcholanthrene, 46 microbial metabolism, 38, 60-61 structure, 32 Micrococcus, in water supply systems, 94 Microcoleus chthonoplastes, PAH metabolism, 37 Microfluorophotometer, 200-201 measurement of fading, 218-219 Micromonospora, from drinking water, 100, 101 Milk, fermentations, 1-2 Molecular biology computing, 194 MOLGEN project, 192 Morarella metabolism of PAH, 35 in water supply systems, 94 Mucor, PAH metabolism, 36
N Naegleria in drinking water, 107 Joiueri, in drinking water, 108
Naphthalene bacterial oxidation, pathway, 42-43 biodegradation, in natural habitats, 63 fungal oxidation, pathway, 44 microbial metabolism, 38, 41-46 structure, 32 Naphthalene dioxygenase, 41-43 Nauicula, PAH metabolism, 37 Nematodes, in drinking water distribution systems, 108 Neurospora crassu benzo[u]pyrene hydroxylase, 53 PAH metabolism, 36 I-Nitropyrene, microbial metabolism, 62 Nitzschiu, PAH metabolism, 37 Nocardia anthracene metabolism, 48 from drinking water, 100, 101 metabolism of PAH, 35 phenanthrene metabolism, 49 Norwalk virus, 133 Nostic, PAH metabolism, 37
0 Oscillutoria naphthalene metabolism, 45-46 PAH metabolism, 37, 38
P Panaeolus cambodginensis, PAH metabolism, 36 subbalteatus, PAH metabolism, 36 Parvovirus, isoelectric points, 142 Penicillium ch ysogenum, PAH metabolism, 36 from drinking water, 102 notatum methylbenz[a]anthracene metabolism, 58 PAH metabolism, 36 ocho-chloron, PAH metabolism, 36 on pipe surfaces, 103 Perkin-Elmer 650-40 spectrofluorophotometer, 200 calibration, 217 Pestalotia, PAH metabolism, 36
242 Petalonia fascia, PAH metabolism, 37 Petriellidium boydii, from drinking water pipe, 103 Phenanthrene bacterial oxidation, pathways, 49-50 bay-region, 46 biodegradation, 63 fungal oxidation, 50-51 K-region, 46 lack of carcinogenicity, 46-48 low biological activity, 46-48 mammalian metabolism, 46 microbial metabolism, 38, 46-51 structure, 32 Phlyctochytrium, PAH metabolism, 36 Phosphorescence, principle, 211-212 Phycomyces blakesleeanus, PAH metabolism, 36 Phytophthora cinnamoni, PAH metabolism, 36 Planctomyces, in water supply systems, 94 PLASMAP, 184 Polio virus, 157 adsorption, 149 to estuarine sediments, 137 to metal surfaces, 135-136 to minerals and soil, 137 to soil, 143 interaction, on solid surface, 154-155 protein VP4, amino acid sequences, 143 type 1 concentration, 162 disinfection, 152 inactivation protection against, 152 in sediments, 151 interaction with metal oxides, 153 isoelectric points, 142 removal from wastewater, 157 from water, 158 survival in containers, 150 in sediments, 150 type 2, isoelectric points, 142 Polycyclic aromatic hydrocarbons (PAH) activation, 33 algal oxidation, 64 biodegradation, 65 in nature, 62-64
INDEX
in cancer, 31 cyanobacterial metabolism, 64-65 distribution, 32 fungal degradation, 64 mammalian metabolism, pathways, 33-34 microbial metabolism, 31-71 pathways, 34-41 nitro-substituted, microbial metabolism, 61-62 oxidized to dihydrodiols, by microorganisms, 38-41 prosynthesis, 32 sources, 32-33 structures, 31-32 Porphyridium cruentum, PAH metabolism, 37 n-Propyl gallate, to retard fading, 208-209 Protein and Nucleic Acid Data Banks, of National Biomedical Research Foundation, 176 Proteus, in drinking water, 91 public health importance, 98-99 Protozoa in drinking water, 87-88, 107 importance, 108 on drinking water distribution system wall/pipe surfaces, 107-108 resistance to chlorine, 108 Prouidencia, in drinking water, public health importance, 98-99 Pseudomonads anthracene metabolism, 48 phenanthrene metabolism, 49 Pseudonionas aeruginosa metabolism of PAH, 35 in drinking water, puhlic health importance, 99 methylbenz[a]anthracene metabolism, 58 resistance to ozone, 103 desmolyticum, metabolism of PAH, 35 in drinking water, 91 public health importance, 98-99 fluorescens metabolism of PAH, 35, 38 rnildenbergii, metabolism of PAH, 35 naphthalene dioxygenase, 41 naphthalene oxidation, 43-44 phenanthrene metabolism, 49
INDEX
243
putida anthracene metabolism, 48 metabolism of PAH, 35, 38, 41 phenanthrene metabolism, 49 pyocyanea, disinfection, from drinking water, 85 rhodocrous, metabolism of PAH, 35 in water supply systems, 94, 96 Psilocybe spp., PAH metabolism, 36 Pyrenebutyric acid, oxygen quenching, 213214
R Reovirus, 159 adsorption, 146 interaction, on solid surface, 154-155 removal, during sludge treatment, 157 Reovirus 3 adsorption, 143 isoelectric points, 142 Rhinovirus 2, isoelectric points, 142 Rhizophlyctis, PAH metabolism, 36 Rhizopus arrhizus, PAH metabolism, 36 stolonifer, PAH metabolism, 36 Rhodotorula glutinis, from drinking water, 104-105 minuta, on water pipes, 105 rubra, from drinking water, 104-105 Hotavirus, 133 adsorption, activated sludge floes, 157 removal, during sludge treatment, 157 SA-11, removal, from water, 158 survival. 149
s Saccharomyces cerevisiae benzo[a]pyrene hydroxylase, 53, 53-55 PAH metabolism, 36 Salmonella in drinking water, 91 public health importance, 98-99 from pipe sediments, 98 typhi, disinfection, from drinking water, 85
typhimurium, 48 in water supply systems, 94 Saprolegnia parasitica, PAH metabolism, 36 Sarcina, in water supply systems, 94 Scenedesmus, 106 Schiff-type dyes, and mechanism of fluroescence fading, 212-213 Schizothrir, 106 Serratia in drinking water, public health importance, 98-99 in water supply systems, 94 Sewage, treatment, 156 Shope papilloma, isoelectric points, 142 Smallpox, isoelectric points, 142 Smittium culicis, PAH metabolism, 36 culisetae, PAH metabolism, 36 simulii, PAH metabolism, 36 Snails, in drinking water distribution systems, 108 Sodium dithionite, to reduce fading, 208 comparison studies, 217-230 Sordaria fimicola, PAH metabolism, 36 Sponges, in drinking water distribution systems, 108 Sporobolomyces salmonicolor, on water pipes, 105 Sporocybe, from drinking water, 102 Standard Methods for the Examination of Water and Wastewater, 86, 100, 102, 103, 106 viral recovery technique, 89 Standards for the Examination of Water and Wastewater, 104 Staphylococcus aureus in drinking water, public health importance, 98-99 in water supply systems, 96 saprophyticus, in water supply systems, 96 in water supply systems, 94 Stern layer, 134-135 Streptococci, group N, 1 Streptococci, lactic, 1-29 adsorption reactions, 7 changes in resistance to bacteriophages, 5-6 concentrated cultures, 16-18
244
INDEX
interactions with bacteriophages, 1-29 lysogenic and phage-carrying cultures, 13-15 multiple strain starters, 19-20 new strains, 3 phage-insensitive, 3, 22-23, 26 phage-resistant mutants, 20-22, 26 plasmid DNA, 23-25 in phage resistance, 23-25 pseudolysogeny, 14-15 restriction and modification systems, 1112 restriction enzymes in, 12-13 strain rotation, 18-19 transduction in, 15-16 Streptococcus cremris, 1, 5 799, 12 AML, 12 EB7, 6 F, restriction endonuclease, 12 KH, 11, 12 plasmid DNA, 24 restriction/modification system, 25 lysogeny, 13-14 M12R, plasmid DNA, 24 phage receptors, 6 R1, 12 restriction/modification systems, 11 SK11, 7 transduction in, 15 Streptococcus lactis, 1, 5 diacetylactis, 1, 5 phage-resistant mechanism, 25 transduction in, 15 lysogeny in, 13-14 ME2, 7 M L 3 phage receptors, 6 phage-insensitive strain, 22-23 restriction/modification systems, 11 transduction in, 15 Streptomyces, from drinking water, 100, 101 Syncephalastrium, PAH metabolism, 36 Synedra, PAH metabolism, 37
T Thamnidium anamlum, PAH metabolism, 36 Thraustochytrium, PAH metabolism, 36 Toluene dioxygenase, 41
Trichoderma from drinking water, 102 on pipe surfaces, 103
U Uzva fasciata, PAH metabolism, 37 Uronemu, on reservoir surfaces, 108
V Vaccinia, isoelectric points, 142 Van der Waals attraction, 134-135 Van der Waals interactions, quantification, Lifshitz theory, 146-147 Verticillium from drinking water, 102 on pipe surfaces, 103 Virus. See also specific virus adsorption, 141-149 applied aspects, 155-163 effect of nature of surface, 144-147 effect of nature of virus, 141-144 effect of organic matter, 148-149 effect of salts and pH, 147-148 hydrophobic interactions, 142-144 isoelectric points of solids important in, 144-145 isothermal relationships, 136-137 kinetic studies, 137 mechanisms, 134-141 protective effects of, 149-152 to surfaces, 133-168 from suspension, 136 theoretical aspects, 134-141 variables affecting, 141-142 in wastewater treatment, 155-157 concentration from water, filters for, 162 disinfection, 151-152, 163 from drinking water, 81-82 in drinking water, 88-89 importance of, 89-90 in drinking water distribution systems, 88-90 effect of cations, 139-140 effect of chaotropic and antichaotropic agents, 138-140 effect of pH, 139-140
245
INDEX
elution, strain dependence, 161 enteric, 133 in environment, 133 detection, 161-163 inactivation in aquatic environment, 153 by disinfectants, 89-90 by drinking water treatment, 81-82 on metal surfaces, 153-154 in soils, 155 on solid surfaces, 153-155 interactions with adsorbent, 145 isoelectric point, 141-142 migration, in subsurface, 161 particles, colloidal behavior, 134 pathogenic, in groundwater, 160 removal from drinking water, 81-82 retention, by soil, 160-161 surface interactions, hydrophobic effects, 138 survival in aquatic and soil environments, 149151 in soils, 150 transport, in environment, 163 Vorticelkz, on reservoir surfaces, 108
W Wastes, land application of, 160-161 Wastewater adsorption, 159-160 advanced treatment, 157-160 biological treatment, 155-157 coagulation, 157-159 filtration, 159-160 microbial quality, 73 Water. See also Wastewater adsorbents of viruses in, 149 chlorination, 73, 91, 110-111 for reducing algal blooms, 107 systems model, 110-113 for virus inactivation, 81-82 chlorine residual, and bacterial levels, 110-1 13 coagulation, 79-81 disinfection, 79, 79-80 research needs, 113-114 distribution systems, 78-79 dynamics, 109-113
ecological processes, 109-110 microbial aftergrowth, 88-108 drinking adsorption, 159-160 advanced treatment, 157-160 coagulation, 157- 159 disinfection in relation to coliforms, 99 filtration, 159-160 other organisms in, 108 trihalomethane (THM) precursors in, 103, I06 filtration, 79-80 flash chlorination, 81 flocculation, 79-81 flow rates, and bacterial levels, 110-111 microbial quality, 73 historical perspective, 75-76 microbiology, future research, 113-117 pipe surfaces, microbial detachment from, 110 potable biological sedimentation, 82 coagulation and filtration, 83 disinfection, 84-86 indicator bacteria reduction by chemical coagulation and filtration, 83-84 microbiology, 73-132 microstraining, 82 pretreatment, 82 rapid sand filtration, 83 roughing filtration, 82 slow sand filtration, 83 trihalornethane (THM) precursors in,
87 pretreatment, 79-80 quality, measures, 114 sedimentation, 79-81 source, 76-79 microbial quality, indicator organisms, 77-78 quality, 77 storage, 79-80 systems management, bacteria counts as evaluative tools, 99-100 tap, virus concentration from, 162 total organic carbon in, and bacterial numbers, 92 treated, 79-88 treatment and distribution, research needs, 115-117 treatment schemes, 79-81
246
INDEX
treatment systems, sanitary requirements, 77 virus concentration from, microporoils filters fur, 145 Waterborne disease, 73-74, 88, 133, 160 in developing countries, 74 and indicator organisms, 115-116 in United States, 74, 77 Water lice, in drinking water distribution systems, 108
chlorine resistance, 105 in drinking water, 86, 104 importance, 105 on drinking water distribution system wall/pipe surfaces, 104-105 ozone resistance, 105 PAH metabolism, 64
Yersinia enterocolitica, in drinking water, public health importance, 98-99 survival, in water, 78
X Xanthomonas, in water supply systems, 93
Y Yeasts. See also specific species benzo[u]pyrene hydroxylase, 53
2 Zeiss Zonax microscope, 200-201, 215-216 calibration, 216-217 Zyngorhynchus tnoelkri, PAH metabolism, 36
CONTENTS OF PREVIOUS VOLUMES Volume 1
Volume 2
Protected Fermentation
Newer Aspects of Waste Treatment Nandor Porges
Mil& Herold and J a n NeEasek The Mechanism of Penicillin Biosynthesis Arnold L. Demain
Aerosol Samplers
Preservation of Foods and Drugs by Ionizing Radiations
A Commentary on Microbiological Assaying F . Kavanagh
Harold W . Batchelor
W . Dexter Bellamy Application of Membrane Filters The State ofAntibiotics in Plant Disease Cont rol
Richard E h r M Microbial Control Methods in the Brewery Gerhasd J . Hass
David Pramer Microbial Synthesis of Cobamides D. Perlman Factors Affecting the Antimicrobial Activity of Phenols E . 0. Bennett Germfree Animal Techniques and Their Applications
The Production of Amino Acids by Fermentation Processes
Shukuo Kinoshita
Newer Development in Vinegar Manufactures Rudolph J . Allgeier and Frank M . Hilde-
brandt The Microbiological Transformation of Steroids T,H , Stoudt
SYMPOSIUM ON ENGINEERING ADVANCES FERMENTATION PRACTICE
Rheological Properties of Fermentation Broths Fred H. Deindoerfm and John M. West
Continuous Industrial Fermentations Philip Gerhardt and M . C . Bartlett
Fluid Mixing in Fermentation Process The Large-Scale Growth of Higher Fungi Radclijjfe F. Robinson and R . S . Davidson AUTHOR INDEX-SUBJECT
IN
J . Y. Oldshue Scale-Up of Submerged Fermentations W. H . Bartholemew
INDEX
247
248
CONTENTS OF PREVIOUS VOLUMES
Air Sterilization Arthur E . Humphrey Sterilization of Media for Biochemical Processes Lloyd L. Kempe Fermentation Kinetics and Model Processes Fred H . Deindoerfer Continuous Fermentation W . D. Maxon
The Metabolism of Cardiac Lactones by Microorganisms Elwood Titus Intermediary Metabolism and Antibiotic Synthesis 1.D. Bu’Lock Methods for the Determination of Organic Acids A. C . H u h AUTHOR INDEX-SUBJECT
Control Applications in Fermentation George]. Fuld AUTHOR INDEX---SUBJECT INDEX
Volume 3 Preservation of Bacteria by Lyophilization Robert J . Heckly
Sphaerotilus, Its Nature and Economic Significance Norman C . Dondero Large-Scale Use of Animd Cell Cultures Donald J . Merchant and C . Rkhard Eidam Protection against Infection in the Microbiological Laboratory: Devices and Procedures Mark A. Chatigny Oxidation of Aromatic Compounds by Bacteria Martin H . Rogoff
Volume 4 Induced Mutagenesis in the Selection of Microorganisms S. 1. Alikhaniun The Importance of Bacterial Viruses in Industrial Processes, Especially in the Dairy Industry F . J . Babel Applied Microbiology in Animal Nutrition Harlow H . Hall Biological Aspects of Continuous Cultivation of Microorganisms T . Holme Maintenance and Loss in Tissue Culture of Specifc Cell Characteristics Charles C . Morris Submerged Growth of Plant Cells L. G . Nickell AUTHOR INDEX-SUBJECT
Screening for the Biological Characterizations of Antitumor Agents Using Microorganisms Frank M . Schabel, Jr., and Robert F . Pittillo
INDEX
INDEX
Volume 5 Correlations between Microbiological Morphology and the Chemistry of Biocides Adrian Albert
The Classification of Actinomycetes in Relation to Their Antibiotic Activity Elio Baldacci
Generations of Electricity by Microbial Action 1. B. David
249
CONTENTS OF PREVIOUS VOLUMES
Microorganisms and the Molecular Biology of Cancer
G. F . Gause Rapid Microbiological Determinations with Radioisotopes Gilbert V . Leuin The Present Status of the 2,3-Butylene Glycol Fermentation Sterling K . Long and Roger Patrick Aeration in the Laboratory W . R . Lockhart and R . W . Squires Stability and Degeneration of Microbial Cultures on Repeated Transfer
Fritz Reusser
Microbial Formation and Degradation of Minerals Melvin P. Siluermun and Henry L. Ehrlich Enzymes and Their Applications lrwin W . Sizer
A Discussion of the Training of Applied Microbiologists B . W . Koft and Wayne W . Umbreit AUTHOR INDEX-SUBJECT
INDEX
Volume 7 Microbial Carotenogenesis Alex Ciegler Biodegradation: Problems of Molecular Recalcitrance and Microbial Fallibility
Microbiology of Paint Films
M . Alexander
Richard T.Ross The Actinomycetes and Their Antibiotics
Cold Sterilization Techniques John B. Opfell and Curtis E . Miller
Selman A. Waksman Fuse1 Oil A. Dinsmoor Webb andlohn L. lngraham AUTHOR INDEX-SUBJECT
INDEX
Volume 6 Global Impacts of Applied Microbiology: An Appraisal Carl-GiiranHedkn and Mwtimer P . Stan Microbial Processes for Preparation of Radioactive Compounds
D. Perlman, A r i s P. Bayan, and Nancy A. Giuffie Secondary Factors in Fermentation Processes
P. Margalith Nonmedical Uses of Antibiotics Herbert S . Goldberg
Microbial Production of Metal-Organic Compounds and Complexes D.Perlmun Development of Coding Schemes for Microbial Taxonomy S. T. Cowan Effects of Microbes on Germfee Animals
Thomas D. Luckey Uses and Products of Yeasts and Yeast-Like Fungi Walter J . Nickerson and Robert G . Brown Microbial Amylases Walter W . Windish and Nagesh S . Mhatre
The Microbiology of Freeze-Dried Foods Gerald J . S i l o a a n and Samuel A. Goldblith Low-Temperature Microbiology Judith Fawell and A. H . Rose
Microbial Aspects of Water Pollution Control
K. Wuhrmann
AUTHOR I N D E X 4 U B J E C T INDEX
250
CONTENTS OF PREVIOUS VOLUMES
Volume 8 Industrial Fermentations and Their Relations to Regulatory Mechanisms Arnold L. Demain
Antiserum Production in Experimental Animals Richard H . Hyde Microbial Models of Tumor Metabolism G. F. Cause
Genetics in Applied Microbiology S. G . Bradley
Cellulose and Cellulolysis Brigitta Norkrans
Microbial Ecology and Applied Microbiology Thomas D. Brock
Microbiological Aspects of the Formation and Degradation of Cellulose Fibers L. Juradek, J . Ross Coluin, and D. R . Whitaker
The Emlogical Approach to the Study of Activated Sludge Wesley 0. Pipes Control of Bacteria in Nondomestic Water Supplies Cecil W . Chambers and Norman A . Clarke The Presence of Human Enteric Viruses in Sewage and Their Removal by Conventional Sewage Treatment Methods Stephen Alan Kollins
Malo-Lactic Fermentation Ralph E . Kunkee INDEX
Volume 10
Media and Methods for Isolation and Enumeration of the E n t e r m m i Paul A. Hartman, George W . Reinbold, and Deui S. Saraswat Crystal-Forming Bacteria as Insect Pathogens Martin H . Rogoff Mycotoxins in Feeds and Foods Emanuel Borker, Nina F . lnsalata, Colette P. Leui, and John S. Witwmun INDEX
Volume 9 The Inclusion of Antimicrobial Agents in Pharmaceutical Products A . D. Russell, June Jenkins, and 1. H. Harrison
Bulking of Activated Sludge Wesley 0. Pipes
AUTHOR INDEX-SUBJECT
Oral Microbiology Heiner Hoffman
AUTHOR INDEX-SUBJECT
The Biotransformation of Lignin to HumusFacts and Postulates R. T.Oglesby, R. F . Christman, and C . H . Driver
Detection of Life in Soil on Earth and Other Planets, Introductory Remarks Rob& L. Starkey For What Shall We Search? Allan H . Brown Relevance of Soil Microbiology to Search for Life on Other Planets G . Stotzky Experiments and Instrumentation for Extraterrestrial Life Detection Gilbert V. k o i n Halophilic Bacteria D. J . Kushner Applied Significance of Polyvalent Bacteriophages S. G . Bradley
25 1
CONTENTS OF PREVIOUS VOLUMES
Proteins and Enzymes as Taxonomic Tools Edward D. Garber and John W . Rippon
Ergot Alkaloid Fermentations William]. Kelleher
Mycotoxins Alex Ciegler and Eioind B . Lillehoj
The Microbiology of the Hen’s Egg R. G . Board
Transformation of Organic Compounds by Fungal Spores Claude Vizina, S. N . Sehgal, and Kamur Singh
Training for the Biochemical Industries I . L. Hepner
Microbial Interactions in Continuous Culture Henry R. Bungay, 111 and Mary Lou Bunf3Y
Volume 12
Chemical Sterilizers (Chemosterilizers) Paul M . Borick Antibiotics in the Control of Plant Pathogens M . J . Thirumalachar AUTHOR INDEX-SUBJECT
AUTHOR INDEX-SUBJECT
INDEX
History of the Development of a School of Biochemistry in the Faculty of Technology, University of Manchester Thomas Kennedy Walker Fermentation Processes Employed in Vitamin C Synthesis Milod Kulhanek
INDEX
CUMULATIVE AUTHOR INDEX-CUMULATIVE TITLEINDEX
Flavor and Microorganisms P. Margalith and Y. Schwurtz
Volume 11
Mechanisms of Thermal Injury in Nonsporulating Bacteria M . C . Allwood and A. D . Russell
Successes and Failures in the Search for Antibiotics Selman A . Waksman
Collection of Microbial Cells Daniel 1. C . Wang and Anthony]. Sinskey
Structure-Activity Relationships of Semisynthetic Penicillins K . E . Price Resistance to Antimicrobial Agents J . S . Kiser, G . 0. Gale, and G . A. Kemp Micromonospora Taxonomy George Luedemann
Fermentor Design R. Steel and T. L. Miller The Occurrence, Chemistry and Toxicology of the Microbial Peptide-Lactones A. Taylor Microbial Metabolites as Potentially Useful Pharmacologically Active Agents D. Perlman and G. P. Peruzzotti
Dental Caries and Periodontal Disease Considered as Infectious Diseases William Gold
AUTHOR INDEX-SUBJECT
The Recovery and Purification of Biochemicals Victor H . Edwards
Chemotaxonomic Relationships Among the Basidiomycetes Robert G. Benedict
INDEX
Volume 13
252
CONTENTS OF PREVIOUS VOLUMES
Proton Magnetic Resonance SpectroscopyAn Aid in Identification and Chemotaxonomy of Yeasts P. A. 1. Gorin a n d ] . F. T. Spencer
Mathematical Models for Fermentation Processes A. G . Frederickson, R. D. Megee, Ill, and H. M . Tsuchija
Large-Scale Cultivation of Mammalian Cells R. C . Telling and P. J. Radlett
AUTHOR INDEX-SUBJECT
INDEX
Volume 14 Large-Scale Bacteriophage Production K . Sargent Microorganisms as Potential Sources of Food /nanendra K. Bhattacharjee Structure-Activity Relationships among Semisynthetic Cephalosporins M . L. Sassiver and Arthur Lewis Structure-Activity Relationships in the Tetracycline Series Robert K. Blackwood and Arthur R . English Microbial Production of Phenazines I . M. lngram and A. C . Blackwood The Gibberellin Fermentation E . G. Jeffreys Metabolism of Acylanilide Herbicides Richard Bartho and David Pramer Therapeutic Dentrifrices 1. K. Peterson Some Contributions of the U.S. Department of Agriculture to the Fermentation Industry George E . Ward Microbiological Patents in International Litigation John V . Whittenburg Industrial Applications of Continuous Culture: Pharmaceutical Products and Other Products and Processes R. C. Righelato and R. Elsworth
Development of the Fermentation Industries in Great Britain John J . H. Hastings Chemical Composition as a Criterion in the Classification of Actinomycetes H. A. Lecheoalier, M a y P. Lechevalier, and Nancy N. Gerber Prevalence and Distribution of AntibioticProducing Actinomycetes ]ohn N . Porter Biochemical Activities of Nocardia R . L. Raymond and V . W . lamison Microbial Transformations of Antibiotics Oldrich K. Sebek and D. Perlmun
In Vioo Evaluation of Antibacterial Chemotherapeutic Substances A. Kathrine Miller Modification of Lincomycin Barney 1. Magerlein Fermentation Equipment G. L. S o l m n s The Extracellular Accumulation of Metabolic Products hy Hydrocarbon-Degrading Microorganisms Bernard]. Abbott and William E. Gledhill AUTHOR INDEX-SUBJECT
INDEX
Volume 15 Medical Applications of Microbial Enzymes Irwin W . S b e r
,
253
CONTENTS O F PREVIOUS VOLUMES
Immobilized Enzymes K. L. Smiley and G . W . Strandberg
Intestinal Microbial Flora of the Pig R. Kenwurthy
Microbial Rennets Joseph L. Sardinas
Antimycin A., a Piscicidal Antibiotic Robert E . Lennon and Claude Vizina
Volatile Aroma Components of Wines and Other Fermented Beverages A . Dinsmoor W e b b and Carlos]. Muller
Ochratoxins Kenneth L. Applegate and John R. Chipley
Correlative Microbiological Assays Ladislav J . Hatika
Cultivation of Animal Cells in Chemically Defined Media, A Review Kiyoshi Higuchi
Insect Tissue Culture W . F. Hink Metabolites from Animal and Plant Cell Culture Irving S.Johnson and George B. Boder Structure-Activity Relationships in Cournermycins John C. Godfrey and Kenneth E . Price Chloramphenicol Vedpal S . Malik Microbial Utilization of Methanol eharles L. Cooney and Daoid W . Levine Modeling of Growth Processes with Two Liquid Phases: A Review of Drop Phenomena, Mixing and Growth P. S. Shah, L. T . Fan, 1. C . Kao, and L. R. Erickson Microbiology and Fermentations in the Prairie Regional Laboratory of the National Research Council of Canada 1946-1971
R. H. Haskins
Genetic and Phenetic Classification of Bacteria R. R. Colwell Mutation and the Production of Secondary Metabolites Arnold L. Demuin Structure-Activity Relationships in the Actinomycins Johannes Meienhofer and Eric Atherton Development of Applied Microbiology at the University of Wisconsin William B. Sarles AUTHOR INDEX-SUBJECT
INDEX
Volume 17 Education and Training in Applied Microbiology Wayne W. Umbreit Antimetabolites from Microorganisms David L. Pruess and James P. Scannell
Volume 16
Lipid Composition as a Guide to the Classification of Bacteria N o m a n Shaw
Public Health Significance of Feeding Low Levels of Antibiotics to Animals Thomas H.Jukes
Fungal Sterols and the Mode of Action of the Polyene Antibiotics J. M. T. Hamilton-Miller
AUTHOR INDEX-SUBJECT
INDEX
254
CONTENTS OF PREVIOUS VOLUMES
Methods of Numerical Taxonomy for Various Genera of Yeasts 2 . Campbell
Recent Developments of Antibiotic Research and Classification of Antibiotics According to Chemical Structure
lanos B k d y Microbiology and Biochemistry of Soy Sauce Fermentation F. M . Young and B. J . B . Wood
SUBJECT INDEX
Volume 19 Contemporary Thoughts on Aspects of Applied Microbiology P . S . S. Dawson and K . L. Phillips
Culture Collections and Patent Depositions T . G . Pridham and C . W . Hesseltine
Some Thoughts on the Microbiological Aspects of Brewing and Other Industries Utilizing Yeast G . G. Stewart
Production of the Same Antibiotics by Members of Different Genera of Microorganisms
Linear Alkylbenzene Sulfonate: Biodegradation and Aquatic Interactions William E . Gledhill
Antibiotic-Producing Fungi: Current Status of Nomenclature C . W . Hesseltine and J . J . Ellis
The Story of the American Type Culture Collection-Its History and Development (1899-1973) William A. Clark and Dorothy H.Geary
Significance of Nucleic Acid Hybridization to Systematics of Actinomycetes S . G.Bradley
Microbial Penicillin Acylases E . J . Vandamme and J . P. Voets SUBJECT INDEX
Volume 18 Microbial Foundation of Environmental Pollutants
Martin Alexander Microbial Transformation of Pesticides
Jean-Marc Bollag
Hubert A . Lccheualier
Current Status of Nomenclature of AntibioticProducing Bacteria
Erwin F. Lessel Microorganisms in Patent Disclosures
Irving Marcus Microbiological Control of Plant Pathogens Y. Henis and 1. Chet Microbiology of Municipal Solid Waste Composting Melvin S. Finstein and M m y L. Morris
Taxonomic Criteria for Mymbacteria and Nocardiae S . G. Bradley and 1. S. Bond
Nitrification and Dentrification Processes Related to Waste Water Treatment D . D. Focht and A. C . Chang
Effect of Structural Modifications on the Biological Properties of Aminoglycoside Antibiotics Containing 2-Deoxystreptamine Kenneth E . Price, John C . Godfrey, and
The Fermentation Pilot Plant and Its Aims D. 1. D. Hockenhull
Hiroshi Kawaguchi
The Microbial Production of Nucleic AcidRelated Compounds
Koichi Ogata
CONTENTS O F PREVIOUS VOLUMES
255
Synthesis of L-Tyrosine-Related Amino Acids by /3-Tyrosinase Hideaki Yamada and Hidehiko Kumagai
Cytotoxic and Antitumor Antibiotics Produced by Microorganisms J . Fuska and B . Proksa
Effects of Toxicants on the Morphology and Fine Structure of Fungi Donald V . Richmond
SUBJECT INDEX
SUBJECT INDEX
Volume 20 The Current Status of Pertussis Vaccine: An Overview Charles R . Manclnrk Biologically Active Components and Properties of Bordetella pertussis Stephen 1. Morse Role of the Genetics and Physiology of Bordetella pertussis in the Production of Vaccine and the Study of Host-Party Relationships in Pertussis Charlotte Parker
Volume 21 Production of Polyene Macrolide Antibiotics Juan F . Martin and Lloyd E. McDaniel Use of Antibiotics in Agriculture Tomomusa Misato, Keido KO, and Isamu Yamaguchi Enzymes Involved in P-Imtam Antibiotic Biosynthesis E. J. Vandamme Information Control in Fermentation Developmen t D . J. D . Hockenhull Single-Cell Protein Production by Photosynthetic Bacteria R . H. Shipmun, L. T . Fan, and 1. C . Kao
Problems Associated with the Development and Clinical Testing of an Improved Pertussis Vaccine George R. Anderson
Environmental Transformation of Alkylated and Inorganic Forms of Certain Metals jitendra Saxena and Philip H. Howard
Problems Associated with the Control Testing of Pertussis Vaccine Jack Cameron
Bacterial Neuraminidase and Altered Immunological Behavior of Treated Mammalian Cells Prasanta K. Ray
Vinegar: Its History and Development Hubert A . Canner and Rudolph J . Allgeier Microbial Rennets M . Sternberg
Pharmacologically Active Compounds from Microbial Origin Hewitt W. Matthews and Barbara Fritche Wade SUBJECT INDEX
Biosynthesis of Cephalosporins Toshihiko Kanzaki and Yukio Fujisawa
Volume 22
Preparation of Pharmaceutical Compounds by Immobilized Enzymes and Cells BernardJ. Abbott
Transformation of Organic Compounds by Immobilized hficrobial Cells lchiro Chtbata and Tetsuya Tosa
256
CONTENTS OF PREVIOUS VOLUMES
Microbial Cleavage of Sterol Side Chains Christoph K . A. Martin
Introduction to Injury and Repair of Microbial Cells F . F. Busta
Zearalenone and Some Derivatives: Production and Biological Activities P. H . Hidy, R. S . Baldwin, R. L. Greasham, C. L. Keith, a n d ] . R. McMulkn
Injury and Recovery of Yeasts and Mold K. E. Stevenson and T . R. Graumlich
Mode of Action of Mycotoxins and Related Compounds F. S . Chu
Injury and Repair of Cram-Negative Bacteria, with Special Consideration of the Involvement of the Cytoplasmic Membrane L . R. Beuchat
Some Aspects of the Microbial Production of Biotin
Yoshikazu lzumi and Koichi Ogata Polyether Antibiotics: Versatile Carboxylic Acid Ionophores Produced by Streptomyces
1.W . Westley The Microbiology of Aquatic Oil Spills R. Bartha and R. M . Atlas
Heat Injury of Bacterial Spores Daniel M . Adorns The Involvement of Nucleic Acids in Bacterial Injury M . D. Pierson, R. F. Coinez, and S. E .
Martin SUBJECTINDEX
Volume 24 Comparative Technical and Economic Aspects of Single-Cell Protein Processes John H . Litchfeld SUBJECT INDEX
Volume 23 Biology of Bacillus popillhe Lee A. BuUQ, ]r., Ralph N . Costilow, and Eugene S . Sharpe Production of Microbial Polysaccharides M . E. Slodki and M . C . Cadmus Effects of Cadmium on the Biota: Influence of Environmental Factors H. Babich and G. Stotzky Microbial Utilization of Straw (A Review)
Youn W . Han
Preservation of Microorganisms
Robert]. Heckly Streptococcus mutans Dextransucrase: A Review
Thomas I . Montville, Charles L. Cooney and Anthony]. Sinskey Microbiology of Activated Sludge Bulking Wesley 0.Pipes Mixed Cultures in Industrial Fermentation Processes David E. F. Hawison Utilization of Methanol by Yeasts
Yoshiki Tani, Nobuo Kato, and Hideaki Yamuda
The Slow-Growing Pigmented Water Bacteria: Problems and Sources Uoyd G. Herman
Recent Chemical Studies on Peptide Antibiotics Iun’ichi S hoji
The Biodegration of Polyethylene Glycols
The CBS Fungus Collection 1. A. Von A m and M . A. A. Schipper
Donald P. Cox
CONTENTS OF PREVIOUS VOLUMES
Microbiology and Biochemistry of Oil-Palm Wine Nduka Okajor Bacterial-Amylases M . B. lngle and R . J . Erickson SUBJECT INDEX
Volume 25 Introduction to Extracellular Enzymes: Frmn the Ribosome to the Market Place R u d y ] . Wodzinski
257
Volume 26 Microbial oxidation of Gaseous Hydrocarbons Ching-Tsang Hou Ecology and Diversity of Methylotrophic Organisms R. S. Hanson Epoxidation and Ketone Formation by C1Utilizing Microbes Ching-Tsang Hou, Ramesh N . Patel, and Allen 1. Laskin
Applications of Microbial Enzymes in Food Systems and in Biotechnology Matthew]. Taylor and Tom Richardson
Oxidation of Hydrocarbns by Methane Monooxygenases from a Variety of Microbes Howard Dalton
Molecular Biology of Extracellular Enzymes Robert F . Ramaley
Propane Utilization of Microorganisms Jerome]. Perry
Increasing Yields of Extracellular Enzymes Douglas E. Eveleigh and Bland S . Montenecourt
Production of Intracellular and Extracellular Protein from n-Butane by Pseudomnas hutanooora sp. nov. Joji Takahashi
Regulation of Chorismate-Derived Antibiotic Production Vedpal S. Malik Structure-Activity Relationships in Fusidic Acid-Type Antibiotics W . o m Daehne, W . 0. Godtfiedsen, and P. R . Ramussen Antibiotic Tolerance in Producer Organisms Leo C . Vining Microbial Models for Drug Metabolism John P. Rosazza and R o b d V . Smith
Effects of Microwave Irradiation on Microorganisms John R. Chipley Ethanol Production by Fermentation: An Alternative Liquid Fuel N . Kosaric, D. C . M. Ng, I . Russell, and G. C. Stewart Surface-Active Compounds from Microorganisms D. 6. Cooper and J . E . Zajic INDEX
Plant Cell Cultures, a Potential Source of Pharmaceuticals W. G. W . Kurz and F . Constabel Bacteriophages of the Genus Clostridium Seicja Ogata and Motoyoshi Hongo SUBJECT INDEX
Volume 27 Recombinant DNA Technology Vedpal Singh Malik Nisin A . Hurst
258
CONTENTS OF PREVIOUS VOLUMES
The Coumermycins: Developments in the Late 1970s John C . Godfrey
Solid Substrate Fermentations
Instrumentation for Process Control in Cell Culture
Microbiology and Biochemistry of Miso (Soy Paste) Fermentation Suinbo H . Abiose, M . C . Allan, and B . 1. B .
Robert 1. Fleischaker, James C. Weaoer, and Anthony J . Sinskey Rdpid Counting Methods for Coliform Bacteria A. hf. Cundell Training in Microbiology University-Bloomington L . s. McCIung
at
Indiana
K. E . Aidoo, R. H e n d y , and B . J . B. Wood
Wood IMIXX
Volume 29 Stabilization of Enzymes against Thermal Inactivation
Alexander M . Klibanov
INDEX
Production of Flavor Microorganisms G.M . Kempler
Volume 28
New Perspectives on Aflatoxin Biosynthesis J. W . Bennett and Siegfried B. Christensen
Immol)ilized Plant Cells P. Hmdelius and K . Mosbach Genetics arid Biochemistry of Secondary Metabolism
Vedpal Singh Malik Partition Afinity Ligand Assay (PALA): Applications in the Analysis of Haptens, Macrotnolecules, and Cells
Bo Mattiasson, Matts Ratnstorp. and TorbjOrn G. I . Ling Accumulation, Metabolism, and Effects of Organophosphorus Insecticides on Microorganisms
Rup La1
Compounds
by
Biofilms and Microbial Fouling W . G. Characklis and K. E . Cooksey Microbial Inulinases: Fermentation Process, Properties, and Applications Erick 1. Vandamine and Dirk G. Derycke Enumeration of Indicator Bacteria Exposed to Chlorine Gordon A. McFeter.s and Anne K. Camper Toxicity of Nickel to Microbes: Environmental Aspects H . Babich and G. Stotzky INDEX