Date of Award

8-1-1983

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Biology

Abstract

Chlamydia trachomatis is an ob ligate in tra c e llu la r parasite re sponsible for a number o f human in fection s. The growing prevalence o f chlamydial in fection s has increased the demand fo r diagnostic testin g fo r th is organism. This study was performed to determine an e ffe c tiv e and p ra ctica l method fo r the diagnostic iso la tio n o f _C. trachomatis in this laboratory.

McCoy and L-929 c e ll monolayers were compared fo r th eir suscep t ib ilit y to C,. trachomatis in fectio n . In a prelim inary study, the number of chlamydial inclusions counted was fiv e -fo ld higher in McCoy c e lls than L-929 c e lls .

McCoy c e ll suspension cultures were tested as a means of sim pli fyin g tissue culture iso la tio n o f J2. trachomatis by elim inating the need fo r pre-formed confluent c e ll monolayers. The recovery of (1. trachomatis in suspended c e lls was comparable to the is o la tio n rates in monolayer cultures in three o f fiv e tr ia ls , but the effectiven ess of the suspension cultures was severely decreased by the presence of b a cteria l contamination and toxic products.

Recovery rates o f CL trachomatis in McCoy c e ll monolayer cul tures were compared with centrifugation performed fo r one hour at 500 x g at 36°C, and 900 x g at room temperature. Although the number o f chlamydial inclusions enumerated a fte r centrifu gation at 500 x g at 36°C was almost double the number recovered with c e n trif ugal forces at 900 x g at room temperature, at 36°C the temperature within the centrifuge could not be con trolled. For subsequent studies, centrifu gation o f the chlamydial inoculum was carried out fo r one hour at 900 x g at room temperature.

The follow in g media were evaluated to find the optimal conditions fo r transport, storage, and inoculation of McCoy c e ll monolayer cultures: sucrose-phosphate medium (2SP), sucrose-phosphate-glutamate medium (SPG), Hanks' balanced sa lt solution, T-soy broth, 2SP contain ing 10% dimethyl su lfoxide, and 2SP containing 10% glycero l. 2SP me dium was determined to be superior to the other media tested for storage o f _C. trachom atis, and as an inoculum-suspending medium. The recovery rates of J2. trachomatis stored in 2SP medium decreased by approximately 20% per day at 4°C, and 50% a fte r one freeze-thaw cycle at -70°C. No sign ifica n t loss o f v ia b ility o f the organism was detected a fte r prolonged storage at -70°C in 2SP medium.

Methods fo r the detection o f chlamydial inclusions in McCoy c e ll monolayer cultures were compared to evaluate the effectiven ess of iodine stain ing, immunoperoxidase (PAP) and immunofluorescence (IFA) assays. From 139 c lin ic a l specimens tested in p a ra lle l, iodine staining detected more Chlamydia-p o sitive samples (9%) than the PAP assay (5%). Ten p ositive samples were frozen and tested a second time with iodine stain and the IFA assay. Only eight samples remained p o sitive for chlamydiae with iodine staining and seven o f the eigh t specimens were p o sitive with the IFA assay. Iodine stain ing of cover- s lip cultures was less expensive, less time-consuming, and easier to interpret than eith er the PAP or IFA assay.

Combining the use o f McCoy c e ll monolayer cultures with iodine staining proved to be the most e ffe c tiv e and p ra ctica l method for iso la tio n o f C. trachomatis.

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