Technical method

Technical methodA rapid method for identifyingbacterial enzymes

J. L. MADDOCKS and MARY JO GREENAN KRUFInstitute of Renal Disease, Welsh National School ofMedicine, Royal Infirmary, Cardiff

Biochemical tests for characterizing bacteria are

often time-consuming. They generally involvedetecting one or more bacterial enzyme by usingcolour changes, coagulation, turbidity or gas for-mation as indices of enzyme activity. These visualchanges depend on the conversion of relatively largeamounts of substrate by the enzyme. Qualitativetests for bacterial enzyme activity could be mademuch more rapid by increasing the sensitivity ofdetection of the reaction products by the humaneye.We describe here a simple and rapid method for

detecting bacterial enzymes that depends on therelease of a fluorescent reaction product (methylum-belliferone) from a non-fluorescent substrate (methyl-umbelliferyl substrate).The following substrates were obtained from

Koch-light Laboratories Ltd, Bucks, England.A 4-methylumbelliferyl-f-D-glucopyranoside (for

,B-D-glucosidase)B 4-methylumbelliferyl-fl-D-galactopyranoside (for

,B-D-galactosidase)C 4-methylumbelliferyl-f-D-glucuronide (for fi-

D-glucuronidase)D 4 - methylumbelliferyl - 2 - acetamido - 2 - deoxy -

P-D-glucopyranoside (for N-acetyl-,-D-glucosa-minidase)

E 4 - methylumbelliferyl - a - D - mannopyranoside(for ox-D-mannosidase).

Fifteen ,u mol of each substrate except B (5 ,u mol)were dissolved in 0-2 ml of dimethylsulphoxide, andthe volume of each solution was made up to 10 mlwith phosphate buffered saline pH 7-3 (Dulbecco'A', Oxoid Ltd, London).Two standard bacteria were studied, Pseudomonas

aeruginosa(NCTC 10490)and Escherichia coli(NCTC10418). These were maintained on Dorset egg slopesand cultured on DST agar (Oxoid Ltd, London)and McConkey agar respectively.To test for the presence of a bacterial enzyme, a

sample of a colony was taken with a glass rod andrubbed vigorously onto a Whatman No. 1 qualita-tive filter paper (Balston Ltd, England). The bacterialsmear was then covered with 2 drops (30 drops per

Received for publication 25 March 1975.

Figure Effect of Esch. coli (NCTC 10418) on methylum-belliferyl substrates for: (A) ,B-D-glucosidase, (B) ,B-D-galactosidase, (C) fl-D-glucuronidase, (D) N-acetyl-p-D-glucosaminidase, and (E) cx-D-mannosidase.Cl A to E: controls of methylumbelliferyl substrates onlyC2: control of Esch. coli ± substrate solvent

ml) of substrate, and a further 2 drops of substratewere placed alongside to serve as a control. Anotherbacterial smear covered by 2 drops of the solvent inwhich the substrate was dissolved served as a secondcontrol. This procedure was performed with each ofthe five substrates using Ps. aeruginosa and Esch. coli.After incubation at 37°C for 10 minutes the spots onthe filter paper were covered with 2 drops (30 dropsper ml) of OIN Na OH. Alkali increases the fluores-cence intensity of methylumbelliferone and alsowashes it away from the bacterial smear so that itcan be more easily seen. The spots on the filter paperwere viewed in a dark room under ultraviolet light(366 nm).The results with Esch. coli (NCTC 10418) are

shown in the accompanying figure. There was lightblue fluorescence of methylumbelliferone withsubstrates A, B, and C, indicating that the organismpossessed the enzymes P-D-glucosidase, ,-D-galac-tosidase, and ,B-D-glucuronidase respectively. Theintensity of the fluorescence was in the order'B > C > A. Tests with substrates D and E gave no

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Letters to the Editor

light blue fluorescence and neither did any of thecontrols. The Esch. coli smears gave a light brownfluorescence.With Ps. aeruginosa (NCTC 10490) there was no

light blue fluorescence of methylumbelliferone withany of the substrates A-E, showing that the tests forenzyme activity were negative. However, theorganism had a green fluorescence, presumably dueto fluorescein, and possibly this could have maskedmethylumbelliferone fluorescence.

This simple method for identifying bacterialenzymes can be performed within minutes comparedwith 18 to 24 hours using standard biochemical

Letters to the Editor

tests. Obviously the scope of this method depends onthe availability of suitable substrates. There are,however, 22 derivatives of methylumbelliferoneavailable commercially which are enzyme substrates,and doubtless others could be synthesized shouldthere be a demand for them. We consider that therapidity of this method might be important bothdiagnostically and economically and justifies itsfurther evaluation.

We are grateful to Mr. P. Blake for photography.When this work was carried out M.J.G. was a pupilof Atlantic College, St. Donat's Castle, Glam.

Book reviewsOrientation of Blocks from Cervical Cones The authors have commented as follows:

For many years we have used a methodfor orientation of blocks and sectionsfrom unbisected cervical cones which ismore simple than that outlined by Sanerkinand Fraser (J. clin. Path., 1975, 28, 202).They identify the anterior lip of the cervixby transfixing it with a needle and infil-trating the tissue with Indian ink. Aquicker way, and just as effective, is tomake a superficial incision across theectocervical surface of the anterior lipwith a razor blade. After parallel slicingof the cervix, at right angles to the razorcut, it is easy to identify the cuts on theanterior lip of the cervix in the subse-quent sections.There is an additional procedure which

we find useful. The purpose of the serialblocking of the cervix is to ensure astep-wise examination of the wholespecimen. Therefore it is essential to makesure that the blocks are all orientatedsatisfactorily in the paraffin wax. This isfacilitated by applying a spot of Indianink on the surface of the block oppositeto the side from which sections are to becut. The first spot goes on the roundedsurface of the first block at 9 o'clock. Theonly block which is not examined inserial fashion is the final block from theedge of the cervix at 3 o'clock, which isblocked on the flat surface.

H. G. RICHMOND and W. N. ROSEPathology Department

Raigmore Hospital,Inverness

Making an incision across the anteriorlip of the ectocervix readily comes tomind and was certainly tried out at ourlaboratory at St. David's Hospital beforewe decided to search for a more satis-factory procedure. We found the incisionmethod quite unacceptable because, inour experience, many cones have numer-ous transverse surgical nicks on both lips,and in examining the final sections we

could not be certain which 'nick' was oursand which the surgeon's.Marking the 'upper surface' of tissue

blocks is a routine procedure at mostlaboratories, and certainly at St David'sHospital, to ensure that the correct('lower') surface is trimmed and sectioned.Instead of a spot, however, we use a crosswith Indian ink.

N. G. SANERKIN

Highcroft, Rudry Road,Lisvane, Cardiff

Frontiers of Radiation Therapy andOncology, Vol. 9. The Relationship ofHistology to Cancer Treatment. Proceed-ings of the 9th Annual West Coast CancerSymposium, San Francisco, 1973. Editedby J. M. Vaeth. (Pp. viii + 316; 111 fig-ures; 75 tables. £20-60.) Basel, Munich,Paris, London, New York, and Sydney:S. Karger AG. 1974.

On the whole, pathologists have been slowto integrate modern experimental tech-niques into their field with the aim ofimproving the prognostic and diagnosticaccuracy of histopathology, althoughrecent developments in the lymphomafield, particularly the use of lymphocytesurface markers, show a promising trendin this direction. With this objective inmind and given the considerable develop-ments of modem cell biology it is perhapsa little disappointing to find in this volumesuch acompletedichotomy between experi-mental and routine histopathologicalclinical considerations. Although thedifficulties are not to be minimized, it isclear that careful observation of thebehaviour of untreated and treatedtumours may illuminate histopathologicalinterpretation while, on the other hand,the consideration of histological charac-teristics in the light of the current con-cepts of cell biology, and particularly cellpopulation kinetics, may also be a valu-able exercise as far as assessing the rele-vance of experimental findings to thehuman situation are concerned. In thisbook the first few chapters touch on these

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