Syed J. Khurshid ( Nuclear Chemistry Division, Pakistan Institute of Nuclear Science & Technology, P.O. Nilore, Rawalpindi. )
M. Mubashir A. Khan ( Pakistan Medical Research Council, National Institute of Health, Islamabad. )
B. Shahnaz ( Pakistan Medical Research Council, National Institute of Health, Islamabad. )
The radiometric method has been used to detect the presence of bacteria in serial dilutions of nine bacterial species with and without agitation. It was also found that the detection time is proportional to the dilution showing high sensitivity down to one colony forming unit and the agitation resulted in different manners depending upon bacterial species (JPMA 37 : 34 , 1987).
The automation in thç clinical microbiology has eased the diagnosis and treatment of the patients by providing the information with rapidity, accuracy, and uniformity. The radio-metric methodology is now utilized in variety of microbiological techniques such as to detect bacteria in blood1-5 rapid anti-biotic susceptibility test6-8 detection of bacteria in food9 and cosmetics, detection of white cell metabolism10 and sterility testing of radiopharmaceuticals11-12.This technique was developed and used by NASA to search life on planets (Mars) 13.
The results with the radiometric method of detection of bacteria has been reported by number of workers. Our experience with the automated Bactec 460 (Figure 1),
an instrument using radio-metric procedure for detection of radiolabelled metabolites is reported here.
MATERIAL AND METHOD
In this automated procedure a 3-5m1 sample is incubated at 35.37°C in a sealed rubber septum vial with a liquid14 C-labelled sterilized substrate with an activity of: 2 uCi per vial. If bacteria are present they metabolize carbohydrate or protein, the components of the substrate as energy source, releasing14 co2 by catabolizing glucose or by decarboxylation of aminoacid produced during incubation. The sterile needles of Bactec 460 pierce through rubber septum into the vial above nutritive media and the14 C02 produced during the incubation period is then aspirated from the test vial through sterilizing filter into the ionization chamber, the electrometer present in Bactec unit then measures the current produced in the ionization chamber. This measurement is converted to growth index (GI) reading which is an arbitrary liner scale related to the amount of radioactivity in the ionization chamber. The amount of 14 CO2 liberated is proportional to the amount of the bacterial growth in the nutrient media. A reading of 100 GI corresponds to 0.025 microcurie of14 C. A threshold GI may be set which is usually 30 for aerobic vials and 20 for anaerobic vials, a reading above threshold level indicates the presence of bacteria.
The ionization chamber of Bactec is exhausted through a CO2 trap to prevent release of radioactive material into room air. The model Bactec 460 which we have, allows 60 vials to be placed on it at one time and do sequential testing, printing the data simultaneously, flagging the sample as positive above threshold GI reading, taking only one minute for each sample. The bacterial concentrations of 10,3 102. 10 and 1 of nine species were prepared in peptone water and used for this experiment. These bacterial species namely were Escherichia co/i 0111, Salmonella typhi, Shigella flexnerii, Salmonella paratyphi B, Proteus mirabilis, Pseudomonas peutrifaciens, Pseudomonas aeruginosa, Providencia steuartti, Salmonella enteritidis, Citrobacter freudii and 2 ml of each bacterial concentration injected in 6B aerobic vial containing tryptic soy broth and14 C labelled substrate and incubated at 37°C to find the effect of concentration on detection time. Another set of each species was prepared in duplicate, one set was incubated without agitation and one with agitation at a speed of 200 strokes min1 at 37°C to find the difference between the behaviour of agitated and non-agitated samples. The samples were checked after every 2 hours upto 12 hours and then after 24 hours, 48 hours, 72 hours and 96 hours.
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