Improved Microscopic Detection of Bacteriuria Edward S. Hyman
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ABSTRACT. A simple method is described which permits the microscopic detection of bacteria in sediments of urine and other fluids, including bacteria that have eluded detection by conventional means. The method introduces increased centrifugal force and stepwise chemical fixation and then conventional staining. It is rapid, economical, and suitable for use in a physician's office. Use of this method immediately reveals those bacteria reported as "significant"by the conventional laboratory culture. More importantly, it immediately reveals the presence of bacteria, living or dead, which are missed by the conventional culture and by the conventional Gram staining procedure. These bacteria usually can be grown in special media and they appear to be related to systemic disease as evidenced by the clinical response to appropriate antibiotics.
Key words: microscopy, bacteriuria, occult bacteriuria, microscopic bacteriuria, Gram stain, fixation of sediment
his is a microscopic technic for finding bacteria in urine sediment that is quick enough to be carried out in a physician's office during a patient's visit, yet is sensitive enough to reveal not only bacteria found by the conventional culture, but also to reveal important bacteria not found either by a conventional culture or by a conventional Gram stain. The method is useful for sputum, exudates, aspirates, and other body fluids. In the examination of urine for bacteria, the Gram stain (and microscopy in general) is well known, rapid, and economical. It is commonly used for a presumptive diagnosis of urinary tract infections and it
T
1052-0295/92/6701 001'/$3 OO/O BIOTECHNIC & HISTOCHEMISTRY Copyright 0 1992 by Williams & Wilkins
Volume 67 Number 1
is referenced to the slower, more expen-
sive, and more respected bacterial culture. A major drawback of the Gram stain, besides its inability to provide antibiotic sensitivities, has been the unreliability of fixation of bacteria and other sediment to the slide. Inadequate fixation reduces sensitivity and precludes quantitation. The presently accepted methods of urine culture, standardized following the recommendations of K a s s ( 1956), are useful for the common, usually symptomatic urinary tract infections due to coliform organisms. The relation of microscopy to this reference has been repeatedly reviewed, most recently by Ferry et al. (1990). The staining method reported here is different. Because of increased centrifugation, and particularly because of improvements in the method of fixation of the sediment to the slide, many more bacteria are retained through staining. Bacteria in large numbers are often revealed in sediments which would be considered negative by other methods of staining and negative by conventional culture because microscopy is capable of revealing organisms that are fastidious or dead. This fixation technique allows quantitation when that is desirable. I t makes possible a quick and reliable diagnosis not only of coliform infections, but it also reveals many previously ignored infections due to other bacteria and yeasts. The numbers of bacteria revealed by this method, and particularly the large numbers of Gram positive cocci revealed, make the method appropriate for reopen-
2
ing old but fundamental questions con-
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cerning the clinical significance of bacteria in urine and their significance in the pathogenesis of some systemic diseases.
MATERIALS A N D M E T H O D S All solutions listed are stable for years. They are stored in bulk and dispensed from dropper bottles. These solutions and other materials are arranged in their general order of use. Glacial acetic acid. Centrifuge tubes (about 10 ml) with round bottom. Centrifuge capable of 10,000 X g. VortexRtype shaker. “Green wash.” Add 40 ml of potassium citrate concentrate (see 13) and 9 g of NaCl to 1 1 of distilled water. Add the dye “light green” SF (C.I. 42095, acid green 5, Sigma gL5382) to give it a n identifying color. Add about 0.5 g of sodium azide (NaN3) as a preservative. Pass this solution through a 0.1 to 0.2 pm filter to remove microscopic particles. Buffer at pH 7.4 (uric acid wash). Add 3.4 g of dibasic potassium phosphate (K2HP04)and 7.6 g of NaCl to 1 1 of water. Reduce the pH to about 7.4 with glacial acetic acid. Add about 0.5 g of sodium azide (NaN3) as a preservative. Pass this solution through a 0.1 to 0.2 pm filter to remove microscopic particles. Alternative: Add 3.5 g of THAM (trishydroxymethyl-aminomethane)and 9 g of NaCl to 1 1 of water. Adjust the pH to about 7.4 with glacial acetic acid. Add about 0.5 g of sodium azide (NaN3) as a preservative. Pass this solution through a 0. l to 0.2 pm filter to remove microscopic particles. Clean microslides which have been coated with dilute alcian blue (see 10) and allowed to dry. Lipid-removing wash. Add 1 5 ml of 1, l , l trichloroethane to 500 ml of absolute methanol. Dilute glutaraldehyde-methanol (fixative for sediment). Add 4 ml of 50%
B 10 t ec h nic XC H is t o c t i e nii st ry
aqueous glutaraldehyde to 500 ml of absolute methanol. Dilute alcian blue. Add 20 ml of glacial acetic acid and 3 ml of strong alcian blue (below)to 500 ml of absolute methanol. Strong alcian blue. Add 0.75 g of alcian blue 8GX (C.I. No. 74240) and 15 ml of glacial acetic acid to 500 ml of absolute methanol. This dye is also known as ingrain blue 1 (Sigma Chemical Company’s product No. A5268). Alcian blue should be certified by the Biological Stain Commission. Violet semiaqueous wash Add 36 ml of 0.4 M potassium citrate concentrate (below), 100 ml of absolute methanol, and 540 ml of acetone to 320 ml of distilled water. Add about 15 mg of gentian violet (C.I. 42535 or 42555) or 3 ml of that solution used in the Gram stain to give a n identifying color. Potassium citrate concentrate. Add 130 g of potassium citrate to 1 1 of water and adjust the pH to about 5.4 with glacial acetic acid or citric acid. The procedure may be divided into six successive phases: A. Preparation of the Urine Before Sedimentation Measure pH, sugar, soluble protein, etc., with a dipstick and specific gravity by refractive index prior to altering the pH, or in a separate aliquot of urine. Should the urine be visually clear, then these parameters may be measured in the supernatant. Acidify, if necessary. If the urine is cloudy or becomes cloudy on cooling and the pH is above 6.5, then the cloudiness is likely due to phosphate crystals which will interfere with microscopy. Lower the pH below 6.5 by adding one or two drops of glacial or 50%acetic acid to the centrifuge tube. Phosphates dissolve, but uric acid may precipitate. If cloudiness remains, it is due to other crystals or to sediment such as leuko-
Improved Microscopic Detection of Bacteriuria
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cytes. Other crystals will be removed later (Step C). Leukocytes will not be altered.
B. Sedimentation An 8 or 10 ml sample is centrifuged in a round bottom tube at 10.000 X g for 10 min. The supernatant is easily decanted from the compact sediment. Tilt the tube downward a t about 45” to drain and replace most of the residual supernatant on the wall with a squirt of “green wash” from a dropper bottle. C. Washing the Sediment If Necessary Should the urine contain more t h a n a 30 mg/dl of sugar, then the sugar will redissolve in a n aqueous stain and release sediment. More than 30 mg/dl of soluble protein or crystals of phosphate, urates, or drugs will interfere with staining and tend to obscure bacteria. Should a small aliquot of urine or of its sediment (or of the sediment in a n alternative tube run simultaneously) show microscopic crystals, then eliminate the protein, sugar, and crystals with a wash. Disperse the sediment in about 3 ml of “green wash” by VortexR disruption, centrifuge, and repeat the wash once. Should the crystals be the orange uric acid, then the first wash should be the phosphate or THAM buffer at pH 7.4 (above). Resuspended urate crystals redissolve quickly on slight warming. Calcium oxalate crystals are neither redissolved, nor do they interfere with microscopy. Washed sediment adheres well to a microslide. (N.B., this important step is the only one that significantly prolongs the whole procedure.) D. Mounting the Sediment on a Precoated Glass Slide Coat clean microslides with a few drops of the above dilute alcian blue and let them dry. (The film of a basic dye favors adherence of a negatively charged bacteria and cells to a negatively charged glass; in my experience coating with albumin is not satisfactory.) After replacing the residual supernatant with “green wash” as in B
3
above and resuspending the sediment, decant a small drop of sediment (or transfer 20 pl with a disposable pipette) onto a coated slide and, with the lip of the tube (or of the pipette), spread it over a 1 to 2 cm2 area of the slide. View the wet sediment under the microscope at 100 X to 400 X without a coverslip to see formed elements such as casts and host cells. Then dry the slide thoroughly with a clean 300 W hot air blower. E. Fixation of Sediment to the Slide before Staining
In rapid succession each of the following reagents is applied with a dropper bottle for a few seconds to one or more handheld slides. 1) Rinse with “lipid removing wash.” (This removes semipolar lipids that act like detergents, releasing sediment.) 2) Fix with dilute glutaraldehyde-methanol. 3 ) Apply dilute alcian blue, then strong alcian blue for further fixation. (The stepwise dye-fixative treatment minimizes efflorescence of any acidic mucoproteins present in the sediment.) 4) Apply the violet semiaqueous wash for a few seconds. (This removes those inorganic salts which compete for solvent water with the organic solvents in the mixture. Hydrophilic salts would redissolve in an aqueous stain and contribute to the release of the fixed film). 5) The sediment is ready for staining without drying.
F. Staining A rapid Gram stain h a s been the most useful. However, many other stains may be used, e.g., methylene blue, Ziehl-Neelsen, periodic-Schiff stain, etc. For epifluorescence with acridine orange, more of the alcian blue should be removed by exposure to acidified methanol (about 0.5 ml of concentrated HC1 in 100 ml of methanol) for a few minutes because the blue dye blocks the inciting light. Dry the stained slide.
4
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G. Microscopy View a t 1000 X under immersion oil without a coverslip or with a coverslip mounted with a standard cement. Quantitation can be achieved by simple arithmetic involving the volume of urine represented by the aliquot of sediment, the area of the smear of the sediment on the slide, and the area of a n 1000 x oil immersion field. The bacteria per volume of urine or per time of collection may be estimated, as was first done for blood cells and casts in urine by Addis (1926). RESULTS On gross examination, the chemically fixed stained slide usually shows more sediment than the heat fixed preparation, unless there is more than a trace of protein present. Protein is more likely to confound the microscopy than to help by retaining
Biotechnic & Histochemistry
sediment. The chemically fixed slide may show a bluish tint due to mucoproteins fixed by alcian blue. This is missing in a washed preparation because the soluble mucoproteins are absent. A s compared to customary heat fixation or to simple methanol fixation, the stepwise chemical fixation described here results in much greater retention of sediment on the slide and better preservation of the morphology of that which is retained. Many more bacteria, erythrocytes, leukocytes, host cells lining the urinary tract (urothelial cells), and casts of kidney tubules are found in the chemically fixed and stained sediment of urine. Bacteria are not infrequently found that previously have been lost to microscopy and are not detected in the conventional culture procedures used today. The differences are best described by illustrations. Figure 1 compares a Gram stain of the
Fig. 1. Gram stain of one urine sediment, (A) fixed chemically, (B) fixed by conventional heating, and (C) fixed by further heating. Note the much greater number and better morphology of Gram negative rods in the chemically fixed sediment as compared to the heat fixed. Host cells are better retained and their morphology i s better preserved by the chemical fixation. Further heating results in pyrolysis (C). x 1000.
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Improved Microscopic Detection of Bacteriuria
same urine sediment chemically fixed (Fig. 1A) and heat fixed (Fig. 1B and C). By chemical fixation, there are about 7 X lo6 Gram negative rods per ml of urine. By conventional heat fixation (Fig. lB), there are considerably fewer rods and their morphology is not as uniform. Further heating (Fig. 1C) results in pyrolysis. Figure 2 is a similar comparison using the sediment of abnormal urine containing numerous granular casts. In urine, a cast is formed when excessive plasma protein leaks through the diseased glomeru-
5
lus during ultrafiltration and the protein gels inside a cylindrical kidney tubule (as a mold). Any structure found inside the cast resided in the urine within that kidney tubule when the protein gelled there. In Fig. 2A, chemical fixation retains and preserves the cast and, after staining, shows Gram positive cocci within the cast in a n optical section. This is proof that those Gram positive cocci came from within the kidney. In B, by heat fixation, the casts are hardly recognizable and Gram positive cocci are not seen. By the
Fig. 2. Gram stain of urine sediment containing many casts, chemically fixed (A) compared to conventionally heat fixed (B). In (A) an optical section through a chemically fixed cast shows many Gram positive cocci (e.g., at arrow) within the cast. By conventional heat fixation (B), casts themselves are difficult to identify (arrow) and Gram positive cocci are not found inside the case or on the slide. x 1000. Fig. 3. Cocci that were not revealed by conventional stain or culture. (A) Gram stain of washed, chemically fixed sediment of the urine of a nephrotic child that contained 2.9 g/dl of protein. It shows a pair of Gram positive diplococci (larger arrow) surrounded by a pink capsule (containing protein as well as carbohydrate), a single Gram positive coccus (large arrow), and,particles resembling lysed cocci that are seen in an old culture of cocci (small arrow). O n average, one organism per photographic frame is calculated to reflect about 28,000 per ml of urine. No bacteria are found in a conventional heat fixed Gram stain (not shown) and these cocci did not (and do not) grow in the conventional urine culture. However they can be grown in special media and in my experience they are associated with illness. In (B) a Gram stain of chemically fixed sediment of a catheterized urine from an arthritic woman shows Gram positive cocci (large arrow), the particles resembling lysed cocci as seen in an old culture (small arrow), forms more lysed, and mononuclear cells (large arrow). The host cells and the bacteria are not seen in a conventional heat fixed Gram stain and repeated conventional cultures yielded no growth. The bacteria can be grown in special media and the illness improved upon elimination of the bacteriuria. x 1000.
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6
conventional culture methods used in hospitals today, this urine gave “no growth.” The cocci can be grown in culture (see discussion). The patient improved upon elimination of the cocci by antibiotics. Figure 3 shows Gram positive cocci in the urinary sediment in illnesses in which they are not found by conventional staining or culturing. In Fig. 3A, the washed, chemically fixed, Gram stained urine sediment of a nephrotic boy with 2.9 g/dl proteinuria shows a pair of Gram positive diplococci with a safranin staining capsule (carbohydrate capsule containing protein) and a single Gram positive coccus. Also present are forms resembling the
Biotechnic & Hi s t oc he niis t r y
lysed cocci seen in old cultures. They range from slightly violet to pink and their margins range from sharp to ill-defined. The urine contained about 5,000 cocci per ml. By conventional Gram staining the protein would form a cake that would either release from the slide, occlude the stain, or obscure the cocci. In Fig. 3B, a Gram stain of urine sediment from a woman with osteoarthritis shows preservation of a cluster of mononuclear cells and again shows cocci and what appear to be the lysed cocci. By heat fixation the cellular detail is usually lost and the cocci are found rarely, if a t all. Figure 4 shows very large numbers of
Fig. 4. Gram stains of chemically fixed urine sediments showing great numbers of bacteria. (A) A clean-catch urine from a man with osteoarthritis and asymptomatic bacteriuria showing innumerable Gram negative rods and also a cluster of Gram positive cocci (arrow) in what might have been a cast that disintegrated. The conventional heat fixed Gram stain would likely be positive and the conventional culture would yield the rods in diagnostic numbers, but the cocci would go undetected by either procedure. The arthritis improved upon elimination of both organisms. (B) Gram negative cocci from the sediment of a catheterized urine specimen from a splenectomized woman with unexplained fever without urinary symptoms. The cocci were not found in the routine heat fixed Gram stain, and the conventional hospital culture yielded no growth. She expired. x 1000. Fig. 5. Artifacts consisting of great numbers of particles of varying sizes appearing in the urine sediment. They originate from inadvertent introduction of the sterile (not particle-free) water-dispersible lubricant used in catheterization and they confound microscopy. x 1000.
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Improved Microscopic Detection of Bacteriuria
bacteria in urinary sediment. Figure 4A is the chemically fixed, Gram stained sediment of a routine clean-catch urine from a man with painful osteoarthritis. The Gram negative rods are too numerous to count, but are estimated a t 1 x 10' to 1 X 109/ml. Some of these rods would easily be found in a conventional heat fixed Gram stain and they would be cultured and reported by the routine hospital laboratory. This is the currently accepted asymptomatic but significant bacteriuria. The coexisting Gram positive cocci are usually not found by either conventional staining or culture used in medical laboratories. In this instance the clustering of the cocci suggests that they might have been in a cast that disintegrated. Both organisms can be cultured, and the patient improved upon elimination of both organisms. Figure 4B shows innumerable Gram negative cocci. The specimen showed no growth by the routine hospital procedure. The patient had had a splenectomy a few years before and on this occasion was febrile. She expired. The cocci had lost their ability to retain iodinated crystal violet, i.e., they were no longer Gram positive. They may have been damaged or dead. Figure 5 shows a n unavoidable artifact sometimes seen in this system. It is the sediment of a plug of sterile lubricating jelly (probably cell walls of algae) introduced when the tip of the urethral catheter was plunged into the lubricating jelly prior to catheterizing the patient. Particulate matter from unfiltered wash solutions is similarly seen in microscopy.
DISCUSSION If there are large numbers of Gram positive cocci in urine, some may be seen in a conventional heat fixed slide of the sediment. Even then, they may not grow in the routine culture procedure used in hospitals today. Many years ago the finding of a few Gram positive cocci in the urine sediment of a very sick woman initiated the evolution of this method. The hospital laboratory reported no growth from her urine, but staphylococci grew in media fa-
/
voring fastidious staphylococci and she improved dramatically with antibiotics which eliminated the cocci. Those bacteria, primarily Gram negative rods, which proliferate in urine as a culture medium to reach very high concentrations in the bladder urine, are easily detected by this method, as seen in Fig. 4A and probably in Figs. 1A and B. (About 2.6 per photographic frame calculates to about 100,000 per ml of urine.) They grow readily in the routine hospital culture protocol, yielding more than lo5 cfu/ml, and since K a s s (1956), with minor modifications to lower the concentrations necessary under certain clinical conditions, it has generally been agreed that viable bacteria at such high concentrations are significant as pathogens. A number of authors have suggested modifying the technique of Gram staining in a n attempt to obtain results as close as possible to those obtained by culture. The suggestion is based upon the assumption that the culture reveals the truth. The method reported here frequently reveals bacteria at equally high concentrations (e.g., Fig. 4B and the cocci in Fig. 4A) that do not grow in the routine hospital culture and thus are not found by the conventional methods used today. The bacteria, primarily cocci, that are seen at lower concentrations (as in Figs. 2 and 3 ) could be dismissed as contaminants or saprophytes, as has been done with low counts by culture in the past. However, they are likely to be important. In Fig. 2A, the Gram positive cocci within casts certainly resided within the kidney at one time and thus are not contaminants. Although it is beyond the scope of this paper describing a method, there is good reason to believe that even free Gram positive cocci not in casts, as in Fig. 3 , are also important. The fact that they do not proliferate or even survive in urine or in the routinely used laboratory culture media does not prove that they are not pathogenic. They can almost always be cultured successfully if suitable media and methods are used. The first isolation usually requires special conditions, primarily mi-
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8
croaerobic or anaerobic conditions, as first noted by Rosenow (1914) and as used in part by Rantz (1942)and Marple (1941).A series of such cultures will be reported elsewhere. Important evidence of their significance comes from the patient’s clinical response to elimination of the bacteria by antibiotics. These clinical aspects are outside the scope of this paper and will be reported separately. They are not new to the literature. The last survey of bacteria appearing in urine not limited by the assumption that the bacteria must grow in urine and in routine media, and without the clinical reference to florid pyelonephritis, was that of Rantz (1942), and before him Marple (1941). Each of them made aerobic and anaerobic cultures of catheterized urines from successive patients admitted to the internal medicine wards at Stanford, irrespective of diagnosis. None had urinary tract symptoms. Rantz and Marple found a preponderance of cocci, and, at a n “arbitrary” level of significance of 100 cfu/ml (equivalent to about one organism per 260 round ocular fields using the 1000 x oil objective, which is well below the levels in Figs. 3 and 4), the urines were positive in a variety of illnesses. Today’s array of antibiotics was not available to those authors and no effort was reported to relate the
B iot ec hnic 81 H ist oc h e m ist rv
bacteria to the illness. I have found these previously ignored bacteria in the urine of many patients suffering from diseases whose unestablished cause has been suspected in the past of being bacterial.
ACKNOWLEDGMENT I thank Dr. Quentin B. Deming of the Albert Einstein College of Medicine in New York for many years of encouragement and for confirmation both of this method and of many of the clinical results referred to here. REFERENCES Addis, T. 1926. The number of formed elements in the urinary sediment of normal individuals. J. Clin. Invest. 2: 409-415. Ferry, S., Andersson, S.-0.. Burman, L. G. and Westman, G. 1990. Optimized urinary microscopy for assessment of bacteriuria in primary care. J. Fam. Pract. 31: 153-161. K a s s , E. 13. 1956. Asymptomatic infections of the urinary tract. Trans. Assoc. Am. Physicians 69: 56-64. Marple. C. D. 1941. The frequency and character of urinary tract infections in a n unselected group of women. Ann. Intern. Med. 14: 2220-2239. Rantz, L. A. 1942. Infections of the urinary tract. In: Advances in Internal Medicine. Chicago, Year Book Medical Publishers, 1: 137-167. Rosenow, E. C. 1914. The newer bacteriology of various infections as determined by special methods. J. Am. Med. Assoc. 63: 903-908.
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3525 Prytania Street, Suite 220, New Orleans, Louisiana 701 15
ABSTRACT. A simple method is described which permits the microscopic detection of bacteria in sediments of urine and other fluids, including bacteria that have eluded detection by conventional means. The method introduces increased centrifugal force and stepwise chemical fixation and then conventional staining. It is rapid, economical, and suitable for use in a physician's office. Use of this method immediately reveals those bacteria reported as "significant"by the conventional laboratory culture. More importantly, it immediately reveals the presence of bacteria, living or dead, which are missed by the conventional culture and by the conventional Gram staining procedure. These bacteria usually can be grown in special media and they appear to be related to systemic disease as evidenced by the clinical response to appropriate antibiotics.
Key words: microscopy, bacteriuria, occult bacteriuria, microscopic bacteriuria, Gram stain, fixation of sediment
his is a microscopic technic for finding bacteria in urine sediment that is quick enough to be carried out in a physician's office during a patient's visit, yet is sensitive enough to reveal not only bacteria found by the conventional culture, but also to reveal important bacteria not found either by a conventional culture or by a conventional Gram stain. The method is useful for sputum, exudates, aspirates, and other body fluids. In the examination of urine for bacteria, the Gram stain (and microscopy in general) is well known, rapid, and economical. It is commonly used for a presumptive diagnosis of urinary tract infections and it
T
1052-0295/92/6701 001'/$3 OO/O BIOTECHNIC & HISTOCHEMISTRY Copyright 0 1992 by Williams & Wilkins
Volume 67 Number 1
is referenced to the slower, more expen-
sive, and more respected bacterial culture. A major drawback of the Gram stain, besides its inability to provide antibiotic sensitivities, has been the unreliability of fixation of bacteria and other sediment to the slide. Inadequate fixation reduces sensitivity and precludes quantitation. The presently accepted methods of urine culture, standardized following the recommendations of K a s s ( 1956), are useful for the common, usually symptomatic urinary tract infections due to coliform organisms. The relation of microscopy to this reference has been repeatedly reviewed, most recently by Ferry et al. (1990). The staining method reported here is different. Because of increased centrifugation, and particularly because of improvements in the method of fixation of the sediment to the slide, many more bacteria are retained through staining. Bacteria in large numbers are often revealed in sediments which would be considered negative by other methods of staining and negative by conventional culture because microscopy is capable of revealing organisms that are fastidious or dead. This fixation technique allows quantitation when that is desirable. I t makes possible a quick and reliable diagnosis not only of coliform infections, but it also reveals many previously ignored infections due to other bacteria and yeasts. The numbers of bacteria revealed by this method, and particularly the large numbers of Gram positive cocci revealed, make the method appropriate for reopen-
2
ing old but fundamental questions con-
Biotech Histochem Downloaded from informahealthcare.com by University of Sydney on 12/29/14 For personal use only.
cerning the clinical significance of bacteria in urine and their significance in the pathogenesis of some systemic diseases.
MATERIALS A N D M E T H O D S All solutions listed are stable for years. They are stored in bulk and dispensed from dropper bottles. These solutions and other materials are arranged in their general order of use. Glacial acetic acid. Centrifuge tubes (about 10 ml) with round bottom. Centrifuge capable of 10,000 X g. VortexRtype shaker. “Green wash.” Add 40 ml of potassium citrate concentrate (see 13) and 9 g of NaCl to 1 1 of distilled water. Add the dye “light green” SF (C.I. 42095, acid green 5, Sigma gL5382) to give it a n identifying color. Add about 0.5 g of sodium azide (NaN3) as a preservative. Pass this solution through a 0.1 to 0.2 pm filter to remove microscopic particles. Buffer at pH 7.4 (uric acid wash). Add 3.4 g of dibasic potassium phosphate (K2HP04)and 7.6 g of NaCl to 1 1 of water. Reduce the pH to about 7.4 with glacial acetic acid. Add about 0.5 g of sodium azide (NaN3) as a preservative. Pass this solution through a 0.1 to 0.2 pm filter to remove microscopic particles. Alternative: Add 3.5 g of THAM (trishydroxymethyl-aminomethane)and 9 g of NaCl to 1 1 of water. Adjust the pH to about 7.4 with glacial acetic acid. Add about 0.5 g of sodium azide (NaN3) as a preservative. Pass this solution through a 0. l to 0.2 pm filter to remove microscopic particles. Clean microslides which have been coated with dilute alcian blue (see 10) and allowed to dry. Lipid-removing wash. Add 1 5 ml of 1, l , l trichloroethane to 500 ml of absolute methanol. Dilute glutaraldehyde-methanol (fixative for sediment). Add 4 ml of 50%
B 10 t ec h nic XC H is t o c t i e nii st ry
aqueous glutaraldehyde to 500 ml of absolute methanol. Dilute alcian blue. Add 20 ml of glacial acetic acid and 3 ml of strong alcian blue (below)to 500 ml of absolute methanol. Strong alcian blue. Add 0.75 g of alcian blue 8GX (C.I. No. 74240) and 15 ml of glacial acetic acid to 500 ml of absolute methanol. This dye is also known as ingrain blue 1 (Sigma Chemical Company’s product No. A5268). Alcian blue should be certified by the Biological Stain Commission. Violet semiaqueous wash Add 36 ml of 0.4 M potassium citrate concentrate (below), 100 ml of absolute methanol, and 540 ml of acetone to 320 ml of distilled water. Add about 15 mg of gentian violet (C.I. 42535 or 42555) or 3 ml of that solution used in the Gram stain to give a n identifying color. Potassium citrate concentrate. Add 130 g of potassium citrate to 1 1 of water and adjust the pH to about 5.4 with glacial acetic acid or citric acid. The procedure may be divided into six successive phases: A. Preparation of the Urine Before Sedimentation Measure pH, sugar, soluble protein, etc., with a dipstick and specific gravity by refractive index prior to altering the pH, or in a separate aliquot of urine. Should the urine be visually clear, then these parameters may be measured in the supernatant. Acidify, if necessary. If the urine is cloudy or becomes cloudy on cooling and the pH is above 6.5, then the cloudiness is likely due to phosphate crystals which will interfere with microscopy. Lower the pH below 6.5 by adding one or two drops of glacial or 50%acetic acid to the centrifuge tube. Phosphates dissolve, but uric acid may precipitate. If cloudiness remains, it is due to other crystals or to sediment such as leuko-
Improved Microscopic Detection of Bacteriuria
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cytes. Other crystals will be removed later (Step C). Leukocytes will not be altered.
B. Sedimentation An 8 or 10 ml sample is centrifuged in a round bottom tube at 10.000 X g for 10 min. The supernatant is easily decanted from the compact sediment. Tilt the tube downward a t about 45” to drain and replace most of the residual supernatant on the wall with a squirt of “green wash” from a dropper bottle. C. Washing the Sediment If Necessary Should the urine contain more t h a n a 30 mg/dl of sugar, then the sugar will redissolve in a n aqueous stain and release sediment. More than 30 mg/dl of soluble protein or crystals of phosphate, urates, or drugs will interfere with staining and tend to obscure bacteria. Should a small aliquot of urine or of its sediment (or of the sediment in a n alternative tube run simultaneously) show microscopic crystals, then eliminate the protein, sugar, and crystals with a wash. Disperse the sediment in about 3 ml of “green wash” by VortexR disruption, centrifuge, and repeat the wash once. Should the crystals be the orange uric acid, then the first wash should be the phosphate or THAM buffer at pH 7.4 (above). Resuspended urate crystals redissolve quickly on slight warming. Calcium oxalate crystals are neither redissolved, nor do they interfere with microscopy. Washed sediment adheres well to a microslide. (N.B., this important step is the only one that significantly prolongs the whole procedure.) D. Mounting the Sediment on a Precoated Glass Slide Coat clean microslides with a few drops of the above dilute alcian blue and let them dry. (The film of a basic dye favors adherence of a negatively charged bacteria and cells to a negatively charged glass; in my experience coating with albumin is not satisfactory.) After replacing the residual supernatant with “green wash” as in B
3
above and resuspending the sediment, decant a small drop of sediment (or transfer 20 pl with a disposable pipette) onto a coated slide and, with the lip of the tube (or of the pipette), spread it over a 1 to 2 cm2 area of the slide. View the wet sediment under the microscope at 100 X to 400 X without a coverslip to see formed elements such as casts and host cells. Then dry the slide thoroughly with a clean 300 W hot air blower. E. Fixation of Sediment to the Slide before Staining
In rapid succession each of the following reagents is applied with a dropper bottle for a few seconds to one or more handheld slides. 1) Rinse with “lipid removing wash.” (This removes semipolar lipids that act like detergents, releasing sediment.) 2) Fix with dilute glutaraldehyde-methanol. 3 ) Apply dilute alcian blue, then strong alcian blue for further fixation. (The stepwise dye-fixative treatment minimizes efflorescence of any acidic mucoproteins present in the sediment.) 4) Apply the violet semiaqueous wash for a few seconds. (This removes those inorganic salts which compete for solvent water with the organic solvents in the mixture. Hydrophilic salts would redissolve in an aqueous stain and contribute to the release of the fixed film). 5) The sediment is ready for staining without drying.
F. Staining A rapid Gram stain h a s been the most useful. However, many other stains may be used, e.g., methylene blue, Ziehl-Neelsen, periodic-Schiff stain, etc. For epifluorescence with acridine orange, more of the alcian blue should be removed by exposure to acidified methanol (about 0.5 ml of concentrated HC1 in 100 ml of methanol) for a few minutes because the blue dye blocks the inciting light. Dry the stained slide.
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G. Microscopy View a t 1000 X under immersion oil without a coverslip or with a coverslip mounted with a standard cement. Quantitation can be achieved by simple arithmetic involving the volume of urine represented by the aliquot of sediment, the area of the smear of the sediment on the slide, and the area of a n 1000 x oil immersion field. The bacteria per volume of urine or per time of collection may be estimated, as was first done for blood cells and casts in urine by Addis (1926). RESULTS On gross examination, the chemically fixed stained slide usually shows more sediment than the heat fixed preparation, unless there is more than a trace of protein present. Protein is more likely to confound the microscopy than to help by retaining
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sediment. The chemically fixed slide may show a bluish tint due to mucoproteins fixed by alcian blue. This is missing in a washed preparation because the soluble mucoproteins are absent. A s compared to customary heat fixation or to simple methanol fixation, the stepwise chemical fixation described here results in much greater retention of sediment on the slide and better preservation of the morphology of that which is retained. Many more bacteria, erythrocytes, leukocytes, host cells lining the urinary tract (urothelial cells), and casts of kidney tubules are found in the chemically fixed and stained sediment of urine. Bacteria are not infrequently found that previously have been lost to microscopy and are not detected in the conventional culture procedures used today. The differences are best described by illustrations. Figure 1 compares a Gram stain of the
Fig. 1. Gram stain of one urine sediment, (A) fixed chemically, (B) fixed by conventional heating, and (C) fixed by further heating. Note the much greater number and better morphology of Gram negative rods in the chemically fixed sediment as compared to the heat fixed. Host cells are better retained and their morphology i s better preserved by the chemical fixation. Further heating results in pyrolysis (C). x 1000.
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Improved Microscopic Detection of Bacteriuria
same urine sediment chemically fixed (Fig. 1A) and heat fixed (Fig. 1B and C). By chemical fixation, there are about 7 X lo6 Gram negative rods per ml of urine. By conventional heat fixation (Fig. lB), there are considerably fewer rods and their morphology is not as uniform. Further heating (Fig. 1C) results in pyrolysis. Figure 2 is a similar comparison using the sediment of abnormal urine containing numerous granular casts. In urine, a cast is formed when excessive plasma protein leaks through the diseased glomeru-
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lus during ultrafiltration and the protein gels inside a cylindrical kidney tubule (as a mold). Any structure found inside the cast resided in the urine within that kidney tubule when the protein gelled there. In Fig. 2A, chemical fixation retains and preserves the cast and, after staining, shows Gram positive cocci within the cast in a n optical section. This is proof that those Gram positive cocci came from within the kidney. In B, by heat fixation, the casts are hardly recognizable and Gram positive cocci are not seen. By the
Fig. 2. Gram stain of urine sediment containing many casts, chemically fixed (A) compared to conventionally heat fixed (B). In (A) an optical section through a chemically fixed cast shows many Gram positive cocci (e.g., at arrow) within the cast. By conventional heat fixation (B), casts themselves are difficult to identify (arrow) and Gram positive cocci are not found inside the case or on the slide. x 1000. Fig. 3. Cocci that were not revealed by conventional stain or culture. (A) Gram stain of washed, chemically fixed sediment of the urine of a nephrotic child that contained 2.9 g/dl of protein. It shows a pair of Gram positive diplococci (larger arrow) surrounded by a pink capsule (containing protein as well as carbohydrate), a single Gram positive coccus (large arrow), and,particles resembling lysed cocci that are seen in an old culture of cocci (small arrow). O n average, one organism per photographic frame is calculated to reflect about 28,000 per ml of urine. No bacteria are found in a conventional heat fixed Gram stain (not shown) and these cocci did not (and do not) grow in the conventional urine culture. However they can be grown in special media and in my experience they are associated with illness. In (B) a Gram stain of chemically fixed sediment of a catheterized urine from an arthritic woman shows Gram positive cocci (large arrow), the particles resembling lysed cocci as seen in an old culture (small arrow), forms more lysed, and mononuclear cells (large arrow). The host cells and the bacteria are not seen in a conventional heat fixed Gram stain and repeated conventional cultures yielded no growth. The bacteria can be grown in special media and the illness improved upon elimination of the bacteriuria. x 1000.
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conventional culture methods used in hospitals today, this urine gave “no growth.” The cocci can be grown in culture (see discussion). The patient improved upon elimination of the cocci by antibiotics. Figure 3 shows Gram positive cocci in the urinary sediment in illnesses in which they are not found by conventional staining or culturing. In Fig. 3A, the washed, chemically fixed, Gram stained urine sediment of a nephrotic boy with 2.9 g/dl proteinuria shows a pair of Gram positive diplococci with a safranin staining capsule (carbohydrate capsule containing protein) and a single Gram positive coccus. Also present are forms resembling the
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lysed cocci seen in old cultures. They range from slightly violet to pink and their margins range from sharp to ill-defined. The urine contained about 5,000 cocci per ml. By conventional Gram staining the protein would form a cake that would either release from the slide, occlude the stain, or obscure the cocci. In Fig. 3B, a Gram stain of urine sediment from a woman with osteoarthritis shows preservation of a cluster of mononuclear cells and again shows cocci and what appear to be the lysed cocci. By heat fixation the cellular detail is usually lost and the cocci are found rarely, if a t all. Figure 4 shows very large numbers of
Fig. 4. Gram stains of chemically fixed urine sediments showing great numbers of bacteria. (A) A clean-catch urine from a man with osteoarthritis and asymptomatic bacteriuria showing innumerable Gram negative rods and also a cluster of Gram positive cocci (arrow) in what might have been a cast that disintegrated. The conventional heat fixed Gram stain would likely be positive and the conventional culture would yield the rods in diagnostic numbers, but the cocci would go undetected by either procedure. The arthritis improved upon elimination of both organisms. (B) Gram negative cocci from the sediment of a catheterized urine specimen from a splenectomized woman with unexplained fever without urinary symptoms. The cocci were not found in the routine heat fixed Gram stain, and the conventional hospital culture yielded no growth. She expired. x 1000. Fig. 5. Artifacts consisting of great numbers of particles of varying sizes appearing in the urine sediment. They originate from inadvertent introduction of the sterile (not particle-free) water-dispersible lubricant used in catheterization and they confound microscopy. x 1000.
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Improved Microscopic Detection of Bacteriuria
bacteria in urinary sediment. Figure 4A is the chemically fixed, Gram stained sediment of a routine clean-catch urine from a man with painful osteoarthritis. The Gram negative rods are too numerous to count, but are estimated a t 1 x 10' to 1 X 109/ml. Some of these rods would easily be found in a conventional heat fixed Gram stain and they would be cultured and reported by the routine hospital laboratory. This is the currently accepted asymptomatic but significant bacteriuria. The coexisting Gram positive cocci are usually not found by either conventional staining or culture used in medical laboratories. In this instance the clustering of the cocci suggests that they might have been in a cast that disintegrated. Both organisms can be cultured, and the patient improved upon elimination of both organisms. Figure 4B shows innumerable Gram negative cocci. The specimen showed no growth by the routine hospital procedure. The patient had had a splenectomy a few years before and on this occasion was febrile. She expired. The cocci had lost their ability to retain iodinated crystal violet, i.e., they were no longer Gram positive. They may have been damaged or dead. Figure 5 shows a n unavoidable artifact sometimes seen in this system. It is the sediment of a plug of sterile lubricating jelly (probably cell walls of algae) introduced when the tip of the urethral catheter was plunged into the lubricating jelly prior to catheterizing the patient. Particulate matter from unfiltered wash solutions is similarly seen in microscopy.
DISCUSSION If there are large numbers of Gram positive cocci in urine, some may be seen in a conventional heat fixed slide of the sediment. Even then, they may not grow in the routine culture procedure used in hospitals today. Many years ago the finding of a few Gram positive cocci in the urine sediment of a very sick woman initiated the evolution of this method. The hospital laboratory reported no growth from her urine, but staphylococci grew in media fa-
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voring fastidious staphylococci and she improved dramatically with antibiotics which eliminated the cocci. Those bacteria, primarily Gram negative rods, which proliferate in urine as a culture medium to reach very high concentrations in the bladder urine, are easily detected by this method, as seen in Fig. 4A and probably in Figs. 1A and B. (About 2.6 per photographic frame calculates to about 100,000 per ml of urine.) They grow readily in the routine hospital culture protocol, yielding more than lo5 cfu/ml, and since K a s s (1956), with minor modifications to lower the concentrations necessary under certain clinical conditions, it has generally been agreed that viable bacteria at such high concentrations are significant as pathogens. A number of authors have suggested modifying the technique of Gram staining in a n attempt to obtain results as close as possible to those obtained by culture. The suggestion is based upon the assumption that the culture reveals the truth. The method reported here frequently reveals bacteria at equally high concentrations (e.g., Fig. 4B and the cocci in Fig. 4A) that do not grow in the routine hospital culture and thus are not found by the conventional methods used today. The bacteria, primarily cocci, that are seen at lower concentrations (as in Figs. 2 and 3 ) could be dismissed as contaminants or saprophytes, as has been done with low counts by culture in the past. However, they are likely to be important. In Fig. 2A, the Gram positive cocci within casts certainly resided within the kidney at one time and thus are not contaminants. Although it is beyond the scope of this paper describing a method, there is good reason to believe that even free Gram positive cocci not in casts, as in Fig. 3 , are also important. The fact that they do not proliferate or even survive in urine or in the routinely used laboratory culture media does not prove that they are not pathogenic. They can almost always be cultured successfully if suitable media and methods are used. The first isolation usually requires special conditions, primarily mi-
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croaerobic or anaerobic conditions, as first noted by Rosenow (1914) and as used in part by Rantz (1942)and Marple (1941).A series of such cultures will be reported elsewhere. Important evidence of their significance comes from the patient’s clinical response to elimination of the bacteria by antibiotics. These clinical aspects are outside the scope of this paper and will be reported separately. They are not new to the literature. The last survey of bacteria appearing in urine not limited by the assumption that the bacteria must grow in urine and in routine media, and without the clinical reference to florid pyelonephritis, was that of Rantz (1942), and before him Marple (1941). Each of them made aerobic and anaerobic cultures of catheterized urines from successive patients admitted to the internal medicine wards at Stanford, irrespective of diagnosis. None had urinary tract symptoms. Rantz and Marple found a preponderance of cocci, and, at a n “arbitrary” level of significance of 100 cfu/ml (equivalent to about one organism per 260 round ocular fields using the 1000 x oil objective, which is well below the levels in Figs. 3 and 4), the urines were positive in a variety of illnesses. Today’s array of antibiotics was not available to those authors and no effort was reported to relate the
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bacteria to the illness. I have found these previously ignored bacteria in the urine of many patients suffering from diseases whose unestablished cause has been suspected in the past of being bacterial.
ACKNOWLEDGMENT I thank Dr. Quentin B. Deming of the Albert Einstein College of Medicine in New York for many years of encouragement and for confirmation both of this method and of many of the clinical results referred to here. REFERENCES Addis, T. 1926. The number of formed elements in the urinary sediment of normal individuals. J. Clin. Invest. 2: 409-415. Ferry, S., Andersson, S.-0.. Burman, L. G. and Westman, G. 1990. Optimized urinary microscopy for assessment of bacteriuria in primary care. J. Fam. Pract. 31: 153-161. K a s s , E. 13. 1956. Asymptomatic infections of the urinary tract. Trans. Assoc. Am. Physicians 69: 56-64. Marple. C. D. 1941. The frequency and character of urinary tract infections in a n unselected group of women. Ann. Intern. Med. 14: 2220-2239. Rantz, L. A. 1942. Infections of the urinary tract. In: Advances in Internal Medicine. Chicago, Year Book Medical Publishers, 1: 137-167. Rosenow, E. C. 1914. The newer bacteriology of various infections as determined by special methods. J. Am. Med. Assoc. 63: 903-908.
