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Centenary of the Gram-stain
Letters to the E d i t o r
277
References
I. Editorial. A celebration of centenaries. J Infect I983; 7: 95-962. Johne A. Ein zweiffel6ser fall von congenitaler tuberkulose. Fortschr der Med I885; 3: Io8-2o2 (footnote on p. 20o). 3. National Tuberculosis Association. Koch R. The aetiology of tubercolosis. Baltimore: Waverly Press, I932: 24. C e n t e n a r y o f the G r a m - s t a i n
Sir, It was gratifying to find in y o u r Editorial I approval of the Preston and MorreU technique of I962 for achieving reproducible results with the Gram-stain. ~ T w e n t y years later, it is w o r t h recording that attempts at further improvement, b y the use of other reagents or schedules, have not been rewarding. Even so, this leaves us with a continuing p r o b l e m in the interpretation of staining results. As we said originallyfl ' organisms such as certain streptococci and clostridia, which are in any case only weakly Gram-positive, tend to be decolorised to a greater extent than was anticipated, especially in preparations from older cultures '. Yet elementary t e a c h i n g - and most systems of t a x o n o m y - w o u l d have us believe that there is a clear distinction b e t w e e n G r a m - p o s i t i v e and Gram-negative. In the real biological world, however, borderlines are often unclear: h o w deeply red m u s t the phenol-red in urea m e d i u m become, if an organism is to be called urease-positive ? Nevertheless, if a fresh isolate is to be identified by accepted schemes of classification, it is often essential to make a firm decision on its G r a m reaction. Is it positive or negative? A category of Gram-variable, which we unwisely s u p p o r t e d previouslyfl merely creates an additional p r o b l e m b y d e m a n d i n g the definition o f yet another borderline. W h e n we reach maturity, we must be bold enough to make decisions. Sitting on the fence is often unhelpful. ( O f course, if one uses any of the unreliable modifications of the Gram-stain, it m a y be inevitable to sit on the fence or to risk a decision which is liable to be incorrect.) Actually, the borderline b e t w e e n G r a m - p o s i t i v e and G r a m - n e g a t i v e has long been accepted among microbiologists. In a review in ~95z, B a r t h o l o m e w and M i t t w e r 3 n o t e d that, if material is to be called Gram-positive, it m u s t withstand such decolorisation as is enough to render G r a m - n e g a t i v e both neisseriae and the nuclei of animal cells, since these have been ~commonly accepted as being Gram-negative. T h i s is consistent with G r a m ' s original discovery as recorded b y Professor J a c o b s o n 4 in his exciting account of that work, which y o u p u b l i s h e d recently, p n e u m o c o c c i retained the violet dye 'while the tissue and the cell nuclei' were decolorised. In the Preston and Morrell techniquefl a standard degree of decolorisation only just removes the violet dye from neisseriae and the nuclei of tissue cells. Therefore, if the bacterial cells in a film are all decolorised (and appear red after counterstaining), they are Gram-negative. Nevertheless, if some of the cells from a pure culture retain the violet dye, then that organism is Gram-positive. W i t h bacteria near the borderline, such as diphtheria bacilli and m a n y clostridia, only a small p r o p o r t i o n of the cells m a y be v i o l e t - even from an
278
Letters to the Editor
actively g r o w i n g c u l t u r e . E v e n w i t h s t r o n g l y G r a m - p o s i t i v e strains s u c h as m i c r o c o c c i , t h e p r o p o r t i o n o f r e d cells will g r a d u a l l y i n c r e a s e w i t h t h e age o f the c u l t u r e f r o m w h i c h t h e film is m a d e . So the G r a m r e a c t i o n o f a p u r e isolate s h o u l d b e assessed o n a y o u n g c u l t u r e . G r a m - n e g a t i v e m e a n s t h a t all t h e cells a p p e a r red. G r a m - p o s i t i v e m e a n s t h a t s o m e o f t h e cells, n o t n e c e s s a r i l y a m a j o r i t y , a n d r a r e l y all o f t h e m , r e t a i n the violet dye.
Department of Bacteriology and Virology, University Medical School, Manchester M r 3 9 P T
Noel W. Preston
References
I. Editorial. A celebration of centenaries. J Infect r983 ; 7 : 95-96. 2. Preston NW, Morrell A. Reproducible results with the Gram stain. J Pathol Bacteriol I962; 8x : 241-243. 3. Bartholomew JW, Mittwer T. The Gram stain. Bact Rev I952; I6: 1-29. 4. Jacobson W. Gram's discovery of his staining technique. J Infect r983; 7:97 -IoI.
277
References
I. Editorial. A celebration of centenaries. J Infect I983; 7: 95-962. Johne A. Ein zweiffel6ser fall von congenitaler tuberkulose. Fortschr der Med I885; 3: Io8-2o2 (footnote on p. 20o). 3. National Tuberculosis Association. Koch R. The aetiology of tubercolosis. Baltimore: Waverly Press, I932: 24. C e n t e n a r y o f the G r a m - s t a i n
Sir, It was gratifying to find in y o u r Editorial I approval of the Preston and MorreU technique of I962 for achieving reproducible results with the Gram-stain. ~ T w e n t y years later, it is w o r t h recording that attempts at further improvement, b y the use of other reagents or schedules, have not been rewarding. Even so, this leaves us with a continuing p r o b l e m in the interpretation of staining results. As we said originallyfl ' organisms such as certain streptococci and clostridia, which are in any case only weakly Gram-positive, tend to be decolorised to a greater extent than was anticipated, especially in preparations from older cultures '. Yet elementary t e a c h i n g - and most systems of t a x o n o m y - w o u l d have us believe that there is a clear distinction b e t w e e n G r a m - p o s i t i v e and Gram-negative. In the real biological world, however, borderlines are often unclear: h o w deeply red m u s t the phenol-red in urea m e d i u m become, if an organism is to be called urease-positive ? Nevertheless, if a fresh isolate is to be identified by accepted schemes of classification, it is often essential to make a firm decision on its G r a m reaction. Is it positive or negative? A category of Gram-variable, which we unwisely s u p p o r t e d previouslyfl merely creates an additional p r o b l e m b y d e m a n d i n g the definition o f yet another borderline. W h e n we reach maturity, we must be bold enough to make decisions. Sitting on the fence is often unhelpful. ( O f course, if one uses any of the unreliable modifications of the Gram-stain, it m a y be inevitable to sit on the fence or to risk a decision which is liable to be incorrect.) Actually, the borderline b e t w e e n G r a m - p o s i t i v e and G r a m - n e g a t i v e has long been accepted among microbiologists. In a review in ~95z, B a r t h o l o m e w and M i t t w e r 3 n o t e d that, if material is to be called Gram-positive, it m u s t withstand such decolorisation as is enough to render G r a m - n e g a t i v e both neisseriae and the nuclei of animal cells, since these have been ~commonly accepted as being Gram-negative. T h i s is consistent with G r a m ' s original discovery as recorded b y Professor J a c o b s o n 4 in his exciting account of that work, which y o u p u b l i s h e d recently, p n e u m o c o c c i retained the violet dye 'while the tissue and the cell nuclei' were decolorised. In the Preston and Morrell techniquefl a standard degree of decolorisation only just removes the violet dye from neisseriae and the nuclei of tissue cells. Therefore, if the bacterial cells in a film are all decolorised (and appear red after counterstaining), they are Gram-negative. Nevertheless, if some of the cells from a pure culture retain the violet dye, then that organism is Gram-positive. W i t h bacteria near the borderline, such as diphtheria bacilli and m a n y clostridia, only a small p r o p o r t i o n of the cells m a y be v i o l e t - even from an
278
Letters to the Editor
actively g r o w i n g c u l t u r e . E v e n w i t h s t r o n g l y G r a m - p o s i t i v e strains s u c h as m i c r o c o c c i , t h e p r o p o r t i o n o f r e d cells will g r a d u a l l y i n c r e a s e w i t h t h e age o f the c u l t u r e f r o m w h i c h t h e film is m a d e . So the G r a m r e a c t i o n o f a p u r e isolate s h o u l d b e assessed o n a y o u n g c u l t u r e . G r a m - n e g a t i v e m e a n s t h a t all t h e cells a p p e a r red. G r a m - p o s i t i v e m e a n s t h a t s o m e o f t h e cells, n o t n e c e s s a r i l y a m a j o r i t y , a n d r a r e l y all o f t h e m , r e t a i n the violet dye.
Department of Bacteriology and Virology, University Medical School, Manchester M r 3 9 P T
Noel W. Preston
References
I. Editorial. A celebration of centenaries. J Infect r983 ; 7 : 95-96. 2. Preston NW, Morrell A. Reproducible results with the Gram stain. J Pathol Bacteriol I962; 8x : 241-243. 3. Bartholomew JW, Mittwer T. The Gram stain. Bact Rev I952; I6: 1-29. 4. Jacobson W. Gram's discovery of his staining technique. J Infect r983; 7:97 -IoI.