Sterilization of MacConkey agar and CLED medium by γ-radiation

Int. J Appl. Radmt. Lwt. Vol. 34, No. 10, pp. 1441 1443, 1983 Printed in Great Britain. All rights reserved

0021-9681:8353.00+0.00 Copyright t" 1983 Pergamon Press Ltd

Sterilization of MacConkey Agar and CLED Medium by ,/-Radiation B. B O G O K O W S K Y 1, E. E I S E N B E R G 2. and G. A L T M A N N ' tSheba Medical Center, Tel Hashomer and Tel-Aviv University Medical School, Ramat-Aviv, Israel and 2Soreq Nuclear Research Centre, Yavne 70600, Israel

(Received 10 January 1983) MacConkcy agar and Cystine Lactose-Electrolyte-Deficient (CLED) agar, media widely used in the bacteriological laboratory and recommended for the detection of urinary tract infections, were sterilized by 7-radiation at a dose of 1.5 Mrad. Both were moditied and adapted to radiation sterilization by adding sodium thioglycollate as a radioprotectant, and by increasing their indicator content. The media performed well when tested with different Enterobacteria and other micro-organisms. Growth and change of indicator reaction were equal in irradiated and autoclaved culture media. Culture media were also evaluated after storage for one month at room temperature and at 4 C and compared well with freshly autoclaved media.

Introduction MacConkey agar and Cystine-LactoseElectrolyte-Deficient (CI.ED) medium are widely used in the bacteriological laboratory, and are recommended for coating both sides of the dip slide used in kits for detection of bacteriuriaY '2) These kits suffer from a high degree of contamination during production due to a complicated two-stage aseptic filling process. Advantages of radiation sterilization, mentioned already in previous works, t~4~ can be realized in the commercial preparation of such kits. In this study the conventional formulas of the media were adapted to ~,-sterilization, and their performance was tested by using several microorganisms.

Materials and Methods

and sealed in polyethylene bags. Irradiation was carried out 3 h later. No special precautions were taken during the preparation. The glassware and deionized water were not sterile, and the room had no devices to prevent contamination with airborne bacteria. Plates prepared in the laboratory as described above were stored at 4~C or room temperature (20-25 C) and were tested up to 30 days. For evaluation of the sterilizing dose, the medium was irradiated in batches of 5 petri dishes, with doses from 0.5 to 1.5 Mrad in two different experiments. Results were recorded after incubation for 14 days at room temperature (20--25 C ) and at 35 C.

Irradiation Samples were irradiated at room temperature by a U~Co source at a dose rate of 250 krad/h _+ 15~o.

Media

Physical and chemical measurements

Commercial dehydrated MacConkey agar (Difco), and CLED medium (Oxoid), were rehydrated, according to instructions, by heating to boiling in order to dissolve the contents completely, and the pH adjusted to 7.2 and 7.4, respectively. Culture media used as controls were autoclaved, and petri dishes were prepared in a conventional way. MacConkey agar and CLED medium prepared for irradiation were augmented with sodium thioglycollate (Merck) and neutral red (BDH) and bromothymolblue (BDH) respectively. The media were poured into plastic petri dishes, cooled for 30 min with covers removed, closed

The content of bromothymolblue in CLED medium and neutral red in MacConkey agar were determined as follows. 3 mL of the culture media were extracted by 3 successive portions of 10 mL of 95'~o ethanol. The combined extract was centrifuged and evaporated while adding buffer solution of pH 5.5 in the case of neutral red, and pH 8.5 in the case of bromothymolblue determinations. After bringing to volume with the buffer, the optical density was read at 535/~m for neutral red, and 615/~m for bromothylmolblue and the content calculated from the calibration curve. Dosimetry and pH measurements were made as previously described, t-~ Hardness of the agar was checked qualitatively by passing an inoculation needle over the surface.

* To whom correspondence should be addressed.

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B. BfXiOKOWSKYet al. Table 1. Comparison of the growth performance (given as the number of colonies grown after inoculation with 0.001 mL of micro-organism culture diluted to 10 6) of 1.5 Mrad radiation sterilized (R) and heat sterilized (H) ('LED medium as a function of temperature and storage time Stored for 2 days at 21) 25 C Microorganism Klebsiella ,~pc. Serratia marcescens Proteus vulgaris Citrobactor dit'ersu~" Shigella hoydii Acinetobacter cah'oaceticus Sahnonella typhimurium P s e u & m . m a s aeruginosa Staphylococcus aureu.~ Streptococcus [t, calis Candida alhicans

Stored for 30 days at 20-25 C 4 C

Freshly preparcd

1t-

-- R -

i-i

R

H

R

1"I

5 4 3 4 4 2 0 5

8 5 2 6 2 2 3 5

2 8 4 5 3 I 3 5

2 2 2 6 3 1 2 5

~ 4 3 5 6 I I 7

5 2 t) 5 3 0 6 5

3 2 3 4 2 I 2 4

2

1

0

I

I

I

0

3 I

2 1

0 2

0 I

0 I

0 2

I 1

Table 2. Comparison of the growth performance (given as the number of colonies grown aftcr inoculation with 0.001 mL of the microorganism culture diluted to 10 ~) of 1.5 Mrad radiation sterilized (R) and heat sterilized (Hi MacConke~ agar media as a function of temperature and storage time Stored for 2 days at 20 2 5 C Microorganism Ewherichia Coli Klehsiella ,we. Citrohacter dirersus Proteus rulgari.s' Serratia marcescen.s Salmonella typhimurium Shigella hovdii Pseuthnnonas aeruginosa ,4cinelohacter cah'oacelicus

Stored for 30 days at 20-25 C 4 ("

Freshly prepared

H

R

H

R

H

R

II

4 4 4 I 3 3 3 6 I

7 5 2 3 3 2 0 2 l

5 2 5 I 5 2 3 3 5

2 2 5 4 5 2 2 3 5

3 2 4 l 2 2 6 3 3

2 I 3 3 4 4 I 2 2

I

Bacteria

M e d i a were inoculated with eleven different species of micro-organisms which arc listed in Tables 1 and 2. Strains were incubated tbr 18 h at 35 C in Brain Heart Infusion. A p p r o p r i a t e dilutions were m a d e from each culture and plates were inoculated using a 0.001 m L calibrated loop. Size, m o r p h o l o g y a n d n u m b e r of colonies were recorded for irradiated and control plates, after incubation at 35 C for one and two days.

Results and Discussion Sterilization of M a c C o n k e y agar and C L E D medium was achieved with a radiation dose of 0.5 and 1.0 M r a d , respectively. The difference in sterilization efficiency is assumed to be mainly due to growth inhibitory properties of the bile salt c o n t e n t in MacC o n k e y agar. The use of b o t h media in the same dip slide kit for bacteriuria tests, as well as possible higher initial c o n t a m i n a t i o n in commercial production, m a d e it necessary to check p e r f o r m a n c e at the higher dose of 1.5 M r a d for both media. At this dose the two media paled considerably, almost entirely losing their indic a t o r content. Therefore some prcliminary studies were needed to a d a p t them to the new process. The

5 2 6 2 3 3 2

M a c C o n k e y agar finally prepared for radiosterilization and bacteriological evaluation contained 60 mg,.'L of neutral red with the pH adjusted to 7.2; CLED medium contained 40 mg.L brom o t h y m o l b l u e , with the p]! adjusted to 7.4. Both media c o n t a i n e d sodium thioglycollate as a radioprotectant at a level of 0.01 moI.L. The use of a r a d i o p r o t e c t a n t enabled us to reduce the a m o u n t of indicator to be added to preserve the required indicator content after radiosterilization. Thus we prevented the d a n g e r of possible toxic effects which might be present if an excessive a m o u n t of indicator is used. tsl Sodium thioglycollate, k n o w n to be a ntdiop r o t e c t a n t due to its SH group, "~ was preferred to o t h e r agents as it is nontoxic and regularly formulated into bacteriological media to obtain highly reduced conditions. Bacterial spores, which are the only flora remaining after boiling the media, are lcss susceptible to protective influences than vegetative organisms, ~7~ so that addition of a radioprotectivc agent does not significantly reduce the radiation sterilization efficiency of o u r process. H u n d r e d s of plates of both media [brmulated as described a b o v e and irradiated at 1.5 M r a d were l b u n d to be sterile. The content of the indicators in the radiosterilized media was found to be 2 6 m g . L lbr neutral red. and

Radiosterilization of culture media 27 mg..L for bromothymolblue. Measurements of pH values showed a slight, but insignificant, decrease of 0.1 in the pH of both media. Qualitative checking of hardness of the radiosterilized media, made by passing an inoculation needle over the surface, showed no practical difference in comparison to autoclaved media. The growth performance of radiosterilized culture media, compared with autoclaved media over a storage time of 30 days, at room and cold storage temperatures, showed no difference when checked for various microorganisms, for which these media are recommended. Growth for all treatments was confluent with inocula of dilution of 10 ~ and is recorded in Tables I and 2 for a dilution of 10 ~'. All bacteria checked gave the same typical reactions on radiosterilized media as on autoclaved media for the various temperature and storage conditions studied. Radiosterilization of culture media in its final

1447,

packaging eliminates the need for aseptic filling, and may be applied in the production of dip slides containing C L E D medium and MacConkey agar for use in bacteriuria tests.

References I. McAllister T. A. Nephron II, 123 (1973). 2. Craig W. A. and Kunin ('. M..4ppl. Alicrohiol. 23, 91~-~ (1972). 3. Altmann G., FAsenberg E. and Bogokowsky B. Int. J. Appl. Radiat. Isot. 30, 527 [1979). 4. Eisenberg E. Bogokowsky B. and AItmann G. Int. J. Appl. Radiat. l,~ot. 32, 891 {1981 ). 5. Neal J. D. I.ah. Pratt. 478 (1973}. 6. Baeq M. Z. Chemical Protection again,~t I~mizing Radiati,n (Charles C. Thomas, Springtield, IL. 1965L 7. Ilannan R. S. Scienl([ic and 7~'chmdo~tcal Prohh,m.~ hw~Jh'ed #t uxin~ I,nizin~ Radiations /i : the Prescrt'ation ¢~! Food. p. 57 (Her Majesty's Sl:alionar'. Office, L¢~n-

don. 1955).