Table Of ContentBIOCHEMICAL CHARACTERISTICS OF CGAGULASE-NEGATIVE
VARIANTS OBTAINED FROM A COAGULASE-POSITIVE
CULTURE OF MICROCOCCUS PYOGENES VAR. AUREUS
A Thesis
Submitted to the Faculty
of
Purdue U n iv ersity
*>y
Jack Norman Baldwin
In P a rtia l F u lfillm en t of the
Requirements fo r the Degree
of
Doctor of Philosophy
August, 1950
PURDUE UNIVERSITY
THIS IS TO CERTIFY THAT THE THESIS PREPARED UNDER MY SUPERVISION
Jack Norman Baldwin
n-r
entitled BIOCHEMICAL CHARACTERISTICS OF COAGULASE-
NEGATIVE VARIANTS OBTAINED FROM A COAGULASE-POSITIVE
CULTURE OF MICROCOCCUS PYOGENES VAR. AURBCJS.________
COMPLIES WITH THE UNIVERSITY REGULATIONS ON GRADUATION THESES
AND IS APPROVED BY ME AS FUEFTLiJNG THIS PART OF THE REQUIREMENTS
FOR THE DEGREE OF
D octor o f Philosophy
Professor in Charge of Thbbih
of School or Department
TO THE LIBRARIAN:----
TS
THIS THESIS IS NOT TO BE REGARDED AS CONFIDENTIAL..
ACKNOWLEDGMENT
The author is indebted to the Department of B io lo g ic a l
Sciences of Purdue U n iv ersity for research f a c i l i t i e s and to
the Tippecanoe Cancer S ociety for funds rm- de av a ilab le for
this research.
Sincere ap p reciation is here expressed to Dr. H. K offler
for h is invaluable su ggestion s and constant patience.
ta b le o f co n te n ts
Page
ABSTRACT............................................................................................................................. v i i i
INTRODUCTION.................................................................................................................. 1
HISTORICAL........................................................................................................................ 3
EXPERIMENTAL.................................................................................................................. 23
Methods........................................................................................................................ 23
Determ ination of Biochem ical C h a r a c te r istic s 23
Pigm entation.......................................................................................... 23
3tone*s G elatinase R eaction................................................. 23
Brom-thymol-blue R eaction....................................................... 24
C r y sta l-v io le t R e a c t i o n .................................................... 24
Plate H em olysis.................................................................................. 24
Tube H em olysis.................................................................................... 24
Coagulase R eaction......................................................................... 26
Fermentation S tu d ie s.................................................................... 26
Preparation of Stock C ultures.................................................... 27
Seleotion of the Parent S t r a in .................... 29
Survival Curves...................................................................... 31
Light Survival Curves........................................................................ 32
Dark Survival Curves................................................................... 39
Generation Time.................................................................................................. 43
Mutation R ates.................................................................................................... 44
Rate at Which Non-chromogenic Variants Occur 44
"Spontaneous" Mutation Rate................................................. 46
"Zero-point*" Mutation Rate.................................................... 47
"End-point" Mutation Rate...................................................... 48
Rate at Which C oagulase-negative V ariants Occur.. 50
Rate at Which B io tin -req u irin g VariantsO ccu r.... 53
Survival Curves of Non-chromogenic V arian ts...................... 58
Biochemical C h a ra cteristics of V ariants................................. 60
i i i
TABLE OF CONTENTS (Cont.)
Page
N utritional S tu dies........................................................................................ 68
Vitamin, Purine and Pyrim idine Requirements of
V arian ts..................................................................................................... 68
B iotin Growth Curves............................................................................. 74
Pim elic Acid Requirements of V a ria n ts.............................. 75
Reversion Experim ents.................................................................................. 77
Reversion of Chromogenesis............................................................ 77
Reversion of B io tin Requirements............................................ 78
transform ation Experim ent....................................................................... 79
DISCUSSION........................................................................................................................ 83
SUMMARY AND CONCLUSIONS..................................................................................... 86
APPENDIX.............................................................................................................................. 88
REFERENCES........................................................................................................................ 103
LIST OF TABLES
Table Page
1 Some biochem ical c h a r a c te r istic s of four known
toxioogen ic stra in s of Mloroooocus pyogenes
▼ar. aureus............................................................................................. 30
2 E ffect of u ltr a v io le t ra d ia tio n s on a suspension
of Micrococous pyogenes var. aureus 196
(Experiment 1 ) ..................................................................................... 35
m
E ffec t of u ltr a v io le t rad ia tio n s on a suspension
«_/
of M icrococous pyogenes var. aureus 196
(Experiment £ ) .................................................................................... 36
4 E ffect of u ltr a v io le t rad iation s on a suspension
of Micrococous pyogenes var. aureus 196
(ExperimenT"3). . . . . . . . . ..............777777...................... 37
5 E ffeot of u ltr a v io le t rad iation s on a suspension
of Mloroooocus pyogenes var. aureus 196
(Experiment 1, dark curve) ................................... 40
6 E ffect of u ltr a v io le t rad iation s on a suspension
of Mloroooocus pyogenes var. aureus 196
(Experiment 2, dark cu rve)...................................................... 41
7 "Spontaneous" m utation rate for non-chromogenic
v a ria n ts of M icrococous pyogenes var. aureus
196........................ 47
8 "Zero-point" mutation rate for non-chromogenic
v a ria n ts of Micrococous pyogenes var. aureus
196 induced ty six minutes exposure to u ltr a -
v lo le t r a d ia tio n ............................................................................... 48
9 "End-point" m utation rate for non-chromogenic
v a ria n ts of M icrococcus pyogenes var. aureus
196 induced by s ix minutes exposure to u ltr a -
v lo le t r a d ia tio n ............................................................................... 50
10 "Spontaneous" m utation rate and "end-point"
mutation rate of co»gulase-negative v a ria n ts
and M. pyogenes var. aureus 196....................................... 52
11 "Spontaneous" mutation rate for b io tin -req u irin g
v arian ts of M icrococcus pyogenes var. aureus
196.................................................................................................................. 56
v
LIST OF TABLES (Cont.)
Table Page
12 "End-point" mutation rate of b io tin -req u irin g
▼arianta of Micrococous pyogenes var. aureus
196 induced by s'lx minutes exposure to u ltra -
v io le t ra d ia tio n .............................................................................. 56
12 Summary of u ltr a v io le t induced and spontaneous
mutation rates of varian ts of Micrococcus
pyogenes var. aureus................................................................... 57
14 K values for the rate of exponential k illin g of
Mloroooocus pyogenes var. aureus 196, and
four non-chromogenic varian ts by u ltr a v io le t
l i g h t ........................................................................................................... 60
15 Biochem ical ch a ra cteristics of some chromogenic
Iso la te s and non-chromogenic variants of M.
pyogenes var. aureus 196......................................................... 62
16 Some biochem ical reactions of variants iso la ted
from experiments designed to determine the
rates a t which coagulase-negative and b io tin -
d e fio le n t varian ts oocur......................................................... 65
17 Medium for n u tritio n a l stu d ie s................................................. 69
18 Vitamin, purine, and pyrimidine requirements of
non-chrom ogenic,ooagulase-negative v a r ia n ts ... 71
19 Growth of the parent and varian t stra in s of
Micrococous pyogenes var. aureus in a
complete medium and a medium devoid of b io tin . 72
20 R elative rates at which the coagu lase-p ositive
parent and coagulase-negative variants grow
during logarithm ic phase in the presence or
absence of b lo tln ........................................................................... 75
vi
LIST OF FIGURES
Figure Page
1 Light su rvival curves for M. pyogenes var.
aureus exposed to u ltr a v io le t ra d ia tio n s................... 38
2 Comparison of lig h t and dark su rviva l curves of
K. pyogenes var. aureus 196. Broken lin e dark
curve, so lid lin e the lig h t curve................................... 42
3 Survival curves for 4 non-chromogenic, u ltr a v io le t
induced v a rian ts and the parent stra in , Mlcro-
oocous pyogenes var. aureus 196......................................... 59
4 Growth of the coa g u la se-p o sitiv e parent 196, and a
eoagulase-negative variant in the presence or
absence of b io tin .............................................................................. 76
vi i
abstract
A known pathogenic cu lture of Mloroooocus pyogenes Tar.
aureus was exposed to u ltr a v io le t rad ia tio n s in an attem pt to
produce v aria n ts lack in g the in v itr o c h a r a c te r istic s o f the
pathogenic m icrooocci.
The o v e r-a ll procedure was to irradiate a washed suspension
of the parent cu ltu re, which was orange, c o a g u la se -p o sitiv e ,
alpha-hem olytic, produoed a c id from mannitol, and grew w ell in a
medium devoid of b io tin . The irra d iatio n procedure was care
fu lly standardized so as to produce 99.99 per cent k illin g .
The surviving c e lls were screened for "zero-point” and "end
point" v arian ts using lack of chromogenesis as the c r ite r io n of
a varian t. "End-point" rates were determined a ls o using lack of
coagulase production and lack o f capacity to grow in the absence
of b iotin as evidence o f a v a r ia n t. The irra d ia tio n was found
to increase the occurrence of non-chromogenic v a r ia n ts, approxi
mately 6 tim es. The rate of apoearanoe of the v a ria n ts screened
d irectly for coagulase production and the in a b ility to grow in
the absence of b io tin was increased 5 and 9 tim es, resp ec tiv ely .
There was a 2.5 fold increase in the number of "end-point"
variants over the number o f "zero-point" mutants.
One hundred and fourteen non-chromogenic v aria n ts that
showed no color reversion a fte r 16 d a ily tra n sfers on y ea st-
water, v e a l-in fu sio n were tested for ferm entation reactions and
other p h y sio lo g ica l te s ts commonly used to id e n tify pathogenic
m icrococci. A to ta l of 114 varian ts were te ste d , of which 22
v i i i
were found to be coagulase-negative and 82 co a g u la se-p o sitiv e.
One hundred and f i f t y orange str a in s iso la ted at random from
plates streaked in an irradiated suspension of the culture were
tested a lso . A ll 32 of the non-ohromogenio coagulase-negative
variants were sharply d iffe r e n tia te d from the co a g u la se-p o sitiv e
stra in s, both w hite and orange, by th e ir in a b ility to produce
alpha-hem olysin and acid from mannitol an aerob ically. When
tested fo r th e ir a b ility to grow in a medium devoid of b io tin
a sim ilar co rrela tio n was found to e x is t; coagulase-negative
variants reouired b io tin for good growth in a syn th etic medium,
while o o a g u la se-p o sitiv e str a in s grew equally w ell in the
presence or absence o f b io tin in the medium.
Forty-three a d d ition al varian ts te ste d were a lso found to
lack th is same se t of pathogenic c h a r a c te r istic s that Evans
(1948) and Evans and Niven (1950) found to be lacking in micro-
cocci Isolated from natural sources.
U ltra v io let su rvival curves of 4 randomly selected , eoag-
ulase-negative v a ria n ts showed th at these strain s were not
"selected out" due to an increased resistan ce to u ltr a v io le t
radiati ons.
ix
