The turbidimetry of mycobacterial and other bacterial suspensions

215 Pages·016.469 MB·English

by  Fisher, Myron Wolf

NORTHWESTERN UNIVERSITY LIBRARY
Manuscript Theses
Unpublished theses submitted for the Masterfs and Doctor1s
degrees and deposited In the Northwestern University Library are open
for inspection, but are to be used only with due regard to the rights
of the authors. Bibliographical references may be noted, but passages
may be copied only with the permission of the authors, and proper
credit must be given in subsequent written or published work. Exten­
sive copying or publication of the thesis in whole or in part requires
also the consent of the Dean of the Graduate School of Northwestern
University.
Theses may be reproduced on microfilm for use in place of
the manuscript itself provided the rules listed above are strictly
adhered to and the rights of the author are in no way Jeopardized.
This thesis b y . . . , ........ « • • • • • • • • * • • •
has been used by the following persons, whose signatures attest their
acceptance of the above restrictions*
A Library which borrows this thesis for use by its patrons is
expected to secure the signature of each user.
NAME AND ADDRESS DATE
~y4. 't / Is"
fa. _ '/a-S^Vv
1
2?
a 1' *A/»'/
Y & ■-■&m> ytf — i
O-
f
A / or 7 1 ~
a I)f\ • [ h 1 ^ y 7! '' '
1
(jW~f^L 5- n // * , y
ryUU^ - ^
i >vxvv^ " V""

m m w m & m m uwrvBasm
TOE TOOTXM«fH£ OF MfC OMC TERXA L
and orora mcmnmh subpensicms
A I)XSSEBTATXOJf
8UBJCETTED TO THE OBADUATO SCHOOL
XH PARTIAL nVtltXMBM OF TOE
for too dogroo
DOCTOR OF PHILOSOPHY
or- mcxERXOLDoa
By
Myron Wolf Fishor
Ewnoton, Illinois
9mm 1951
ProQuest Number: 10101401
All rights reserved
INFORMATION TO ALL USERS
The quality of this reproduction is dependent upon the quality of the copy submitted.
In the unlikely event that the author did not send a complete manuscript
and there are missing pages, these will be noted. Also, if material had to be removed,
a note will indicate the deletion.
uest,
ProQuest 10101401
Published by ProQuest LLC (2016). Copyright of the Dissertation is held by the Author.
All rights reserved.
This work is protected against unauthorized copying under Title 17, United States Code
Microform Edition © ProQuest LLC.
ProQuest LLC.
789 East Eisenhower Parkway
P.O. Box 1346
Ann Arbor, Ml 48106 - 1346
TABLE OF CONTENTS
Page
Part I.. Introduction. 1
Part IX, Review of the Literature, 4
A. The Development of Turbidimetric
Method® for Quantitative Bacteriology. 4
B. The Dae of Transmitted Light or
Scattered Light for Bacteriological
Turbidimetry, 9
C. The Optical Properties of Bacteria and
Bacterial Suspension® in Relation to
Turbidimetry, 14
D. The Optical Properties of the Suspen­
sion Medium in Relation to
Bacteriological Turbidimetry, 28
E. The Turbidimetric Measurement of the
Growth of Tubercle Bacilli. 30
Part XIX. Statement of the Problem. 37
Part XV, Experimental Methods and Result®. 38
A, Materials, 3B
B, General Methods. 44
C, Specific Experimental Methods and
Results. 52
Part V. Interpretation of Results and Discussion, 103
A. The Optical Properties of the
Suspension Medium in Relation to
Turbidimetry. 103
B. The Optical Properties of Bacterial
?
Cells in Relation to Turbidimetry. 10
C. The Reliability of Turbidimetric
Methods Applied to Bacterial Suspen­
sions Containing Cell Aggregate®, Ill
B. The Measurement of the Growth of
Nondisperse Suspensions of Tubercle
Bacilli by Turbidimetric Methods. 115
TABLE OF CQWKOTS
(continued)
Pago
S, Sha Effect Of tha Initial pH of
Various Hedta on the Growth of H3?Kr. 122
j Part VI. Suamary ana Gonclufllono. 125
Bibliography.
Vita.
X, XHTROBUCTIOSS
On, of the most widely applied procedures in bacteri­
ology ie the turbidimetry of bacterial suspensions for the
purpose of measuring bacterial growth, numbers, or density,
tinder certain conditions, as with homogeneously turbid and
relatively stable cell suspensions, such procedures are
believed to supply objective data which more closely approxi­
mate the actual eonoentration of bacterial substance than that
of any other known method, particularly when photoelectric
instruments are employed* An additional reason for the wide­
spread use of optical methods is that they can be performed
with relative ease, safety, and rapidity, the latter being of
eepeelal importance with bacterial cultures that are capable
of doubling their mass in 20 to 30 minutes, it is possible,
moreover, to conduct growth studies with cultures maintained
in optical cuvettes, thus permitting continuous or periodic
measurements on a single culture so that sampling errors are
minimised and there is little danger of contamination.
On the other hand, there are definite limitations to
the applicability of turbidimetric methods. An outstanding
one is that such procedures do not distinguish between viable
and non-viable cells, a circumstance which is not objection­
able in the usual type of growth study wherein one in concerned
with estimations of the total concentration of bacterial
protoplasm. This is not the case, however, In the standardi­
sation or measurement of suspensions wherein the viable cell
z
content Is of critical importance, such ac in preparations
at BOG vaccines.
Another limltatiea is that only certain types of bact-
•rial suepeniilens «rs oenaidered to be satisfactory for tor*
bldlaetrlo analysis. this has been characterised by the taeit
understanding that "homogeneously tufbid* suspensions oan be
measured with precision* and that an organism which produces
macroscopic eelx aggregates la the form of clumps, flakes*
or granules is not suitable for turbidimetry. fhis has been
particularly true in the ease at the subsurface growth of
tubercle bacilli in media which do not contain certain dis­
persing agents* and has necessitated the development of other
Methods which arc more laborious and possibly less accurate
than the.turbidimetric procedures which are used for other
bacteria, this problem will be discussed In greater detail
in a subsequent section of this thesis.
Finally* it may be stated that the Interpretation of
data furnished by a turbidimetric method is limited by the
Investigator's knowledge of the assumptions on which such
interpretations are based. Although the subsequent literature
survey Indicates that there have been serious efforts made to
define and demonstrate a number of variables involved in the
interpretation of turbidimetric data, important points have
been dealt with only in theory and not by experiment, fhis
thesis ie a report of experiments done to investigate a number
of these theoretical considerations, an well as experiments
done to reinvestigate some factors which have been regarded as
3
being well-established by previous experiaente. In a more
general sense, this is also a study of th« suitability of two
representative photeeleotri© instruments for tho turbidimetry
of baeterial suepenaions under a variety of eemdltlono, lnolud-
Ing relatively non-dlsporse suspensions of tuberele b&cllli.
Although a portion of the subsequent material has been pre­
sented previously by the author (1)» it is being repeated for
the sake of clarity and completeness*
II. m v t m OF fHK LITERATURE
Recording to SO* <2), tho first instrument developed
for quantitative turbidimetric analysis was designed by
Rietearde O) in 109%, Although, this particular instrument
was not employed in bacteriology, its design and method of
operation established a basis for the subsequent use of similar
instruments in bacteriology. The Richards* instrument ®oa~
slated of two upright cylinders with a standard or reference
suspension placed in one. and with a suspension of unknown
density placed in the ether. An empty test tube was fitted
to each cylinder ao that the height of the column of each sus­
pension could be varied. Both the tubes and the cylinders
were made of glass, and the design of this apparatus was such
that suspensions were illuminated by a light eouroe which was
lateral to the long amis of the cylinders. The internal tubes
were manipulated until equal light Intensities were observed
when viewed from above. At this point, the length of each
column of suspension was measured and the ratio of these lengths
was related to the concentration of material in the unknown
suspension. This method was used by Richards for estimating
the concentration of silver bromide in aqueous suspensions.
and he named hie instrument a “nephelometer*, based on the
©reek word anfttlab * cloud.
In 19$0f KX©lmsann ik) described an apparatus basically