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Correlation of resistance to streptomycin with age of the bacterial culture PDF

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Preview Correlation of resistance to streptomycin with age of the bacterial culture

CORRELATION OF RESISTANCE TO STREPTOMYCIN WITH AGE OF THE BACTERIAL CULTURE A Thesis Presented to the F aculty of the Graduate School U niversity of Southern C alifo rn ia In P a rtia l F u lfillm en t of the Requirements fo r the Degree M aster of Science by Marvin Reitman February 1950 UMI Number: EP55016 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. .Oissertaifott PiiblisJ'tiftg UMI EP55016 Published by ProQuest LLC (2014). Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code ProQuest; ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 /3a 'a-e £ 379 This thesis, written by ...........................lARVI^LREITM .N................................... under the guidance of h.i.a„ Faculty Committee, and approved by all its members, has been presented to and accepted by the Council on Graduate Study and Research in partial fulfill­ ment of the requirements for the degree of MASTER OF SCIENCE .............................................. Dean Faculty Committee Chairman ... TABLE OP CONTENTS CHAPTER PACE I . BACKGROUND AND STATEMENT OF PROBLEM.................. 1 I I . EXPERIMENTS.............................. . . .............................. - 4 Appearance of re s is ta n t v a ria n ts in a growing cu ltu re of a se n sitiv e s tra in ............................... 4 Procedure . . . . . ......................................................... 4 R esults and d i s c u s s i o n ......................................... . 5 Growth, p a tte rn of a re s is ta n t s tra in inoculated in to a se n sitiv e c u l t u r e ................................. 11 P ro c e d u re .......................................................................... 11 D iscussion ............................................... 15 E ffect of reducing the proportion of re s is ta n t c e lls on the growth p a tte rn of the re s is ta n t s tra in in a predom inantly se n sitiv e cu ltu re 16 P ro c e d u re .......................................................................... 16 D is c u s s io n ..................................................................... 18 Test to determ ine the production of a d iffu sib le in h ib ito ry substance by the se n sitiv e s tr a in .............................................................. 18 Procedure ............................................................................. 18 D iscussion . . . * ..................................................... 19 I I I . DISCUSSION AND SUMMARY . .............................................. 21 D is c u s s io n ......................... 21 Summary.................................................................................... 22 BIBLIOGRAPHY....................................................................................................... 24 LIST OF TABLES TABLE PAGE I . Number of B acteria R esistant to Varied C oncentrations of Streptom ycin in Samples taken from a B roth G ulture over a F ifty - Hour Period (Using Smooth S tra in ) ..................... 7 A. Humber of S ensitive Organisms to one R esistan t Organism (Per M l.) a t S pecified In te rv a ls (Using Smooth S tra in ) ............................ 7 I I . Number of B acteria R esistan t to V aried C oncentrations of Streptom ycin in Samples Taken from a B roth C ulture over a F ifty - Hour Period (Using Smooth S tra in ) . . . . . 8 B* Number of S en sitiv e Organisms to one R esistan t Organism (Per M l.) at S pecified In te rv a ls (Using Smooth S tra in ) ............................ 8 I I I . Number of B acteria R esistan t to V aried C oncentrations of Streptom ycin in Samples Taken from a Broth C ulture over a F ifty - Hour Period (Using Rough S train ) . . . . . . 9 C. Number of S en sitiv e Organisms to one R esistan t Organism (Per M l.) a t S pecified In te rv a ls (Using Rough S train ) ................................. 9 IV. Number of B acteria R esistant to Varied C oncentrations of Streptom ycin in Samples Taken from a B roth C ulture over a F ifty - Hour Period (Using Rough S train ) . . . . . . 10 D. Number of S en sitiv e Organisms to one R esistan t Organism (Per N l.) a t S pecified In te rv a ls (Using Rough S train ) ............................... 10 V. The E ffect of Increasing the Number of R esistan t C ells I n itia lly Present on the Growth P attern of R esistan t C ells in a Predom inantly S ensitiv e C ulture of the Parent S train . . . . . ............................................... 13 E. Number of S en sitive C ells to One R esistan t C ell (Using Rough S train ) ......................................... 13 iv TABLE PAGE VI. The E ffect of In creasin g the Number of R esistan t C ells I n itia lly P resent on the Growth P a tte rn of R esistan t C ells in a Predom inantly S en sitiv e C ulture of the Parent S train . . . . . . . ..................... . . . .14 F. Number of S en sitiv e C ells to One R esistan t C ell (Using Rough S train ) .......................... 14 V II. R esistan t Organisms Per Ml. B roth C ulture as Determined by P latin g s on Streptom ycin Agar, C ontaining 1.1 U nits Streptom ycin Per Ml. a t S pecified In te rv a ls . . . . . . . . . . . 17 V III. R esistan t Organisms Per Ml. Broth C ulture as Determined by P latin g s on Streptom ycin Agar, Containing 1.1 U nits Streptom ycin Per Ml. at S pecified In te rv a ls 17 LIST OP FIGURES FIGURE PAGE 1. Stepwise M utation to R esistance 6 2. Development of R esistant S train From a R esistan t V ariant in Presence of Streptom ycin . . . . . . . . ............................................ 20 CHAPTER I BACKGROUND AND STATEMENT OF PROBLEM The mechanism which governs h e re d ita ry c h a ra c te ristic s in b a c te ria u su a lly m aintains a re la tiv e ly homogeneous popu­ la tio n under constant c u ltu ra l co n ditio ns. In sp ite of th is , the occurrence of m utants among the c e lls can e a sily be de­ te c te d using media which favor growth of the v arian ts but not of the Mnorm aln c e lls . Using th is technique Ryan and Schneider (1948, 1949) c le a rly dem onstrated the environm ental fa c to rs resp o n sib le fo r the establishm ent of a h istid in e independent s tr a in from a dependent p aren t. Sim ilar rep o rts described the is o la tio n of stra in s showing opposite biochem i­ cal c h a ra c te ris tic s , (Zamenhof, 1946; Englesberg and S ta n ie r, 1949) and extreme d ifferen ces in re sista n c e (M iller and Bohnhoff, 1947; Yegian and Budd, 1948) from th a t of the p a re n t• From th e above work and others (Braun, 1946; Paine and Finland, 1948) i t can be concluded th a t m utations occur re g u la rly in growing cu ltu res and the extent to which the m utants m u ltip ly In the presence of the p arent s tra in is determ ined by the influence of environm ental conditions. The b a c te ria l cu ltu re besides showing g enetic v a ria ­ tio n s, passes through a cycle of gross, non-genetic changes re la tin g to cu ltu re age. These changes asso ciated w ith the 2 p h y sio lo g ical growth phases have been shown fo r m etabolism (Moony and Winslow, 1935), size (Huntington and Winslow, 1937), e le ctro p h o retic p o te n tia l (Moyer, 1936), and heat pro ­ duction (Bayne-Jones and Rhees, 1929). V ariations in the resistan c e of the c u ltu re during the growth phases have been shown fo r sulphanilam ide (Youmans, 1948), p e n ic illin (Bigger, 1944; P ra tt and Dufrenoy, 1948, c ry s ta l v io le t (Hoffman and Rahn, 1944), and other agents (Sherman and Albus, 1923). The in h ib ito ry actio n of streptom ycin, on the other hand, has been shown to be equally e ffe c tiv e on young and re stin g c e lls (Hamre, Rake and Donovick, 1946). I t is evident th a t v a ria tio n s in re sista n c e occur reg u la rly due to rare but constant m utations in growing cu ltu res and cu ltu re age. The purpose of the p resent ex p eri­ ments was to determ ine w hether any re la tio n sh ip could be shown to e x is t between the cu ltu re age and the number of re s is ta n t m utants. N eisseria c a ta rrh a lis A.T.C. #7900 was chosen as the te s t organism because i t is n on-sporulating, does not form chains or clumps and grows w ell a t pH 7 .8, a value a t which streptom ycin is both stab le and a c tiv e . The organism e x ists as a diplococcus and colonies were assumed to re s u lt from ^ in d iv id u al*1 organism s since the morphology remained con­ s ta n t. The smooth s tra in during iso la tio n in sa lin e was converted Into a rough s tra in as judged by its c u ltu ra l 3 appearances. The smooth s tra in produced l i t t l e tu rb id ity in b ro th and gave ris e to convex colonies having an even edge. The rough v a rie ty produced a tu rb id b ro th and colonies pos­ sessed crenated bord ers. Streptom ycin calcium chloride complex was chosen as the agent ag ain st which resistan ce was te s te d due to the s ta b ility of th is a n tib io tic and its im portance in m edical b acterio lo g y .

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