A Rapid Technic for Quantitating Wound Bacterial Count Christopher Magee, BA, Charlottesville, Virginia Beth Haury, MA, Charlottesville, Virginia George Rodeheaver, PhD, Charlottesville, Virginia James Fox, MD, Charlottesville, Virginia Milton T. Edgerton, MD, Charlottesville, Virginia Richard F. Edlich, MD, PhD, Charlottesville, Virginia
bacteriology is now used to determine graft bed receptiveness [I], predict the safety of wound closure [2], and monitor the course of burns [3]. The degree of contamination appears to be directly related to risk of wound sepsis. Wounds containing more than lo5 organisms per gram of tissue run a high risk of infection. A bacterial level of lo5 or lower in wounds indicates that sepsis is unlikely. Quantitation of the number of bacteria in tissue can be done by homogenizing a weighed tissue specimen and performing formal colony counts using standard serial diluting and culturing technics. This type of assay is completed after an 18 to 24 hour incubation period. By this time, the results of the study have an insignificant effect upon surgical decisions. An improved technic [4] has been developed to predict rapidly the critical number of bacteria in wounds. This technic has proved reliable and accurate in our hands for the diagnosis of experimental and clinical infection and is reproducible in other laboratories. Quantitative
Material and Methods Rapid Slide Technic
The rapid slide technic consists of a direct microscopic measurement of the number of bacteria in the tissue homogenate. The specimen weighing approximately 500 mg is obtained utilizing an aseptic technic. The size of the specimen has an important influence on the quantitative results. As the size of the specimen is reduced, the bacterial population is proportionally decreased. We have found that
From the Department of Plastic Surgery, University of Virginia School of Medicine, Charlottesville, Virginia. This work was supported by agrant from the United States ArmyResearch and Development Command, Washington, DC. Reprint requests should be addressed to Richard F. Edlich. MD, Department of Plastic Surgery, University of Virginia School of Medicine, Charlottesville, Virginia 22901. ’ Junior Faculty Clinical Fellow, American Cancer Society.
760
accuracy of the technic is diminished considerably when these measurements are made on specimens less than 500 mg. After the specimen is weighed on an analytical balance, it is homogenized in 5 ml of 0.9 per cent sodium chloride, utilizing a Polytron@ omnimixer (Brinkman Instruments Inc, Westbury, NY). An aliquot of the homogenate is then subjected to serial dilutions. A measured amount of the undiluted homogenate (0.01 ml) and/or a serial dilution is spread uniformly over a delineated 1 cm2 area of a glass slide. The slide is then placed on a warm hotplate until the smear is dried. Fixation of the smear is accomplished by addition of methanol onto the surface of the smear. When the methanol evaporates, the smear is subjected to the improved Gram stain technic developed in our laboratory [S]. The slide is flooded with buffered crystal violet which is allowed to stand for 60 seconds. The primary stain is then poured off the slide with the iodophor mordant. The slide is then flooded with more mordant which remains on the slide for 1 minute. The slide is then decolorized uniformly with 95 per cent ethanol until the solvent flows colorlessly from the slide. Decolorization of the smear usually takes 5 to 10 seconds. Excess alcohol is removed by rinsing the slide with water. The counterstain, safranin, is then added to the slide for 60 seconds before it is washed off with water. Each slide is allowed to dry and is examined under oil immersion using a 100X objective. Ten separate fields of each smear are examined and the average number of bacteria per field is recorded. This number is multiplied by the number of fields in the 1 cm2 area (4,000), giving the total number of bacteria in the 0.01 ml aliquot of the undiluted tissue homogenate. This number is then multiplied by 500 to give the number of bacteria in the original 5 ml sample. (Figure 1.) When the number of bacteria in the smear of the homogenate is too numerous to count, the number of bacteria per field in the first tenfold homogenate is taken into account in the final calculation of the number of bacteria in the undiluted homogenate. The final results are expressed as the number of bacteria per gram of tissue. Design of Study
The purpose of the present study was to examine the reliability and accuracy of the rapid slide technic in
The American Journal of Surgery
Quantitating Bacterial Count
A.
6.
No.
No.
Bacteria in 10 fields -_10
No. -
Bacteria 1 field
bacteria
1field
4000 fields No. =lcm2X yq2c
where I field=
0.025
bacteria
10,
mm2
IO_
10’
IO.
Day 1 No. Bacteria c.
I
homogenate volume (ml.)
bacteria =-
X
-X I 02
0.01 ml.
Sample wt.
(sm.)
gm. tissue
10’
,a*
10’
10.,0’
10’
Day 2
101
(01
10’10’
101
Day 3
to+
,a5
10’
10’
Day 4
Figure 2. Staphylococcus aureus count of wounds as ascertained by rapid slide count and colony count.
Figure 7. Rapid bacterial quant/tation technic.
quantitating the number of bacteria in contaminated wounds in experimental animals. Forty male, albino, Hartley guinea pigs (weighing 300 to 350 gm) were anesthetized with an intraperitoneal injection of sodium pentobarbital. The back of each animal was clipped with electric shears and then swabbed with 70 per cent ethyl alcohol. Using a tuberculin syringe with a 25 gauge needle, two dermal injections were made on each side of the vertebral column, 1.5 cm lateral to the spine. Staphylococcus aureus (12,600) (American Type Culture Collection Center, Rockville, MD) was the organism injected into the dermal tissues in one group of animals, while Escherichia coli was injected into the dermal wounds in the remaining animals. The level of inoculum employed in these studies varied from lo4 to lo7 organisms. In each group, the animals were sacrificed on the first, second, third, and fourth days after inoculation. The dermal injection sites were excised and subjected to quantitative bacteriologic technics [6]. Concomitantly, a clinical study was initiated employing tissue specimens from patients with infections who were admitted to the Plastic Surgical Service of the University of Virginia Medical Center. The reliability and accuracy of the rapid slide technic was determined by comparing the results of the rapid slide technic with those obtained by routine serial dilution and culturing technics. The results of the rapid slide technic were reported to the surgeon within 30 minutes, while the quantitative counts derived from the culture technic were available 18 to 24 hours after culturing the homogenate. Results
The rapid slide technic gave reliable and accurate measurements of the wound bacterial count for wounds containing more than 2 X lo5 organisms per gram of tissue. (Figures 2 and 3.) When the level of contamination was 2 X lo5 organisms per gram of
Volume 133, June 1977
Day 1
-
Colon* Cc.“nl
(1------o
lopidSlid.
Day
2
Cow,
Day
3
Figure 3. Escherichia coli count of wounds as ascertained by rapid slide count and colony count.
tissue or greater, the number of organisms determined by the rapid slide technic did not differ significantly from that determined by the serial dilution and culturing technics. It is fortuitous that lo6 bacteria is the critical number of bacteria that will induce clinical infection in experimental animals and humans. The major advantage of the rapid slide technic is the speed with which the results are available to the surgeon. The culturing technic is obviously a valuable supplement to the rapid slide technic since it provides opportunity for speciation and antibiotic sensitivity testing.
761
Magee et al
When the level of bacteria in a wound biopsy was less than 2 X lo5 organisms per gram of tissue, bacteria usually were not visible on the slide. Failure to identify this low level of inoculum has little clinical significance since these small numbers of bacteria will not result in clinical infection. The rapid slide technic proved reliable for quantitation of gramnegative rods as well as the more easily recognized gram-positive cocci. The age of the wound at the time of biopsy did not alter the accuracy of the rapid slide technic. With serial dilution and culturing technics, eighteen of the thirty clinical specimens exhibited bacterial counts of less than 2 X lo5 organisms per gram of tissue; sixteen (89 per cent) were predicted by the rapid slide technic. Of the twelve samples containing more than 2 X lo5 bacteria, the number of bacteria in the specimen was accurately predicted to within half a logarithm of actual count obtained from nutrient agar plates.
basic changes, our accuracy in differentiating gram-positive from gram-negative organisms has improved considerably. The results of our rapid slide assay are reported as the number of bacteria per gram of tissue. Specifying the exact number of bacteria per gram of tissue allows the physicians to monitor any treatment effects. Demonstration of a reduction in wound bacterial count after initiating a treatment substantiates the benefits of that therapy. It is important to emphasize that the value of the rapid slide technic is realized primarily in the speed with which the results are available to the surgeon. The development of the rapid slide technic does not replace the quantitative serial dilution and plating technics. These latter technics are always performed concomitantly since they allow us to speciate the pathogen and perform antibiotic sensitivity testing. The results of these additional tests are essential to the management of the surgical patient with infection.
Comments
After repeated observations of the clinical significance of the number of bacteria present in tissue, a rapid method of performing quantitative wound cultures was developed by Heggers, Robson, and Doran [4]. Their technic was based on a direct microscopic examination of a measured aliquot of the homogenate. When we employed the technic in our laboratory, two problems were encountered. The technic was not reliable when the tissue sample size was smaller than 0.5 gm. When such minute specimens were studied, the total bacterial count susceptible to quantitation was considerably diminished. This problem was corrected by obtaining tissue samples weighing over 0.5 gm for rapid slide assay. We have established a useful guideline for physicians that allows them to estimate the weight of the specimen: 2 X 1 X 0.5 cm pieces of tissue weigh approximately 0.5 gm. The second major drawback in the rapid slide technic was our failure to differentiate bacteria accurately by the Gram stain technic. In many cases, gram-positive organisms were considered to be gram-negative. The technical factors contributing to the too easy decolorization of the gram-positive organisms were the method of fixation and the composition of the mordant. Heat fixation of the smear has been abandoned in favor of methanol fixation. The unstable mordant, Iz-KI, has been replaced by an iodophor with a long shelf life. With use of these
762
Summary
An improved technic has been developed to predict rapidly the critical number of bacteria in tissues. This technic gave reliable and accurate measurements for wounds containing more than 2 X lo5 organisms per gram of tissue. It is fortuitous that lo6 bacteria is the critical number of bacteria that will induce clinical infection in experimental animals and humans. The rapid slide technic is now being utilized routinely in our medical center to determine graft bed receptiveness, predict the safety of wound closure, and monitor the course of burns.
References 1. Krizek TJ, Robson MC, Kho E: Bacterial growth and skin graft survival. Surg Forum 18: 518, 1967. 2. Robson MC, Duke WF, Krizek TJ: Rapid bacterial screening in the treatment of civilian wounds. J Surg Res 14: 426, 1973. 3. Krizek TJ, Flagg SV, Wolfort FG, Jabaley ME: Delayed primary excision and skin grafting of the burned hand. Plest Recoflstr Surg 51: 524, 1973. 4. Heggers JP, Robson MC, Doran ET: Quantitative assessment of bacterial contamination of open wounds by a slide technique. Trans R Sot Trop Med Hyg 63: 532, 1969. 5. Magee CM, Rodeheaver G, Edgerton MT, Edlich RF: A more reliable Gram staining technic for diagnosis of surgical infections. Am JSurg 130: 341, 1975. 6. Madden J, Edlich RF, Schauerhamer R, Prusak M, Borner J, Wangensteen OH: Application of principles of fluid dynamics to surgical wound irrigation. Curr Top Surg Res 3: 85, 1971.
The American Journal of Surgery
bacteriology is now used to determine graft bed receptiveness [I], predict the safety of wound closure [2], and monitor the course of burns [3]. The degree of contamination appears to be directly related to risk of wound sepsis. Wounds containing more than lo5 organisms per gram of tissue run a high risk of infection. A bacterial level of lo5 or lower in wounds indicates that sepsis is unlikely. Quantitation of the number of bacteria in tissue can be done by homogenizing a weighed tissue specimen and performing formal colony counts using standard serial diluting and culturing technics. This type of assay is completed after an 18 to 24 hour incubation period. By this time, the results of the study have an insignificant effect upon surgical decisions. An improved technic [4] has been developed to predict rapidly the critical number of bacteria in wounds. This technic has proved reliable and accurate in our hands for the diagnosis of experimental and clinical infection and is reproducible in other laboratories. Quantitative
Material and Methods Rapid Slide Technic
The rapid slide technic consists of a direct microscopic measurement of the number of bacteria in the tissue homogenate. The specimen weighing approximately 500 mg is obtained utilizing an aseptic technic. The size of the specimen has an important influence on the quantitative results. As the size of the specimen is reduced, the bacterial population is proportionally decreased. We have found that
From the Department of Plastic Surgery, University of Virginia School of Medicine, Charlottesville, Virginia. This work was supported by agrant from the United States ArmyResearch and Development Command, Washington, DC. Reprint requests should be addressed to Richard F. Edlich. MD, Department of Plastic Surgery, University of Virginia School of Medicine, Charlottesville, Virginia 22901. ’ Junior Faculty Clinical Fellow, American Cancer Society.
760
accuracy of the technic is diminished considerably when these measurements are made on specimens less than 500 mg. After the specimen is weighed on an analytical balance, it is homogenized in 5 ml of 0.9 per cent sodium chloride, utilizing a Polytron@ omnimixer (Brinkman Instruments Inc, Westbury, NY). An aliquot of the homogenate is then subjected to serial dilutions. A measured amount of the undiluted homogenate (0.01 ml) and/or a serial dilution is spread uniformly over a delineated 1 cm2 area of a glass slide. The slide is then placed on a warm hotplate until the smear is dried. Fixation of the smear is accomplished by addition of methanol onto the surface of the smear. When the methanol evaporates, the smear is subjected to the improved Gram stain technic developed in our laboratory [S]. The slide is flooded with buffered crystal violet which is allowed to stand for 60 seconds. The primary stain is then poured off the slide with the iodophor mordant. The slide is then flooded with more mordant which remains on the slide for 1 minute. The slide is then decolorized uniformly with 95 per cent ethanol until the solvent flows colorlessly from the slide. Decolorization of the smear usually takes 5 to 10 seconds. Excess alcohol is removed by rinsing the slide with water. The counterstain, safranin, is then added to the slide for 60 seconds before it is washed off with water. Each slide is allowed to dry and is examined under oil immersion using a 100X objective. Ten separate fields of each smear are examined and the average number of bacteria per field is recorded. This number is multiplied by the number of fields in the 1 cm2 area (4,000), giving the total number of bacteria in the 0.01 ml aliquot of the undiluted tissue homogenate. This number is then multiplied by 500 to give the number of bacteria in the original 5 ml sample. (Figure 1.) When the number of bacteria in the smear of the homogenate is too numerous to count, the number of bacteria per field in the first tenfold homogenate is taken into account in the final calculation of the number of bacteria in the undiluted homogenate. The final results are expressed as the number of bacteria per gram of tissue. Design of Study
The purpose of the present study was to examine the reliability and accuracy of the rapid slide technic in
The American Journal of Surgery
Quantitating Bacterial Count
A.
6.
No.
No.
Bacteria in 10 fields -_10
No. -
Bacteria 1 field
bacteria
1field
4000 fields No. =lcm2X yq2c
where I field=
0.025
bacteria
10,
mm2
IO_
10’
IO.
Day 1 No. Bacteria c.
I
homogenate volume (ml.)
bacteria =-
X
-X I 02
0.01 ml.
Sample wt.
(sm.)
gm. tissue
10’
,a*
10’
10.,0’
10’
Day 2
101
(01
10’10’
101
Day 3
to+
,a5
10’
10’
Day 4
Figure 2. Staphylococcus aureus count of wounds as ascertained by rapid slide count and colony count.
Figure 7. Rapid bacterial quant/tation technic.
quantitating the number of bacteria in contaminated wounds in experimental animals. Forty male, albino, Hartley guinea pigs (weighing 300 to 350 gm) were anesthetized with an intraperitoneal injection of sodium pentobarbital. The back of each animal was clipped with electric shears and then swabbed with 70 per cent ethyl alcohol. Using a tuberculin syringe with a 25 gauge needle, two dermal injections were made on each side of the vertebral column, 1.5 cm lateral to the spine. Staphylococcus aureus (12,600) (American Type Culture Collection Center, Rockville, MD) was the organism injected into the dermal tissues in one group of animals, while Escherichia coli was injected into the dermal wounds in the remaining animals. The level of inoculum employed in these studies varied from lo4 to lo7 organisms. In each group, the animals were sacrificed on the first, second, third, and fourth days after inoculation. The dermal injection sites were excised and subjected to quantitative bacteriologic technics [6]. Concomitantly, a clinical study was initiated employing tissue specimens from patients with infections who were admitted to the Plastic Surgical Service of the University of Virginia Medical Center. The reliability and accuracy of the rapid slide technic was determined by comparing the results of the rapid slide technic with those obtained by routine serial dilution and culturing technics. The results of the rapid slide technic were reported to the surgeon within 30 minutes, while the quantitative counts derived from the culture technic were available 18 to 24 hours after culturing the homogenate. Results
The rapid slide technic gave reliable and accurate measurements of the wound bacterial count for wounds containing more than 2 X lo5 organisms per gram of tissue. (Figures 2 and 3.) When the level of contamination was 2 X lo5 organisms per gram of
Volume 133, June 1977
Day 1
-
Colon* Cc.“nl
(1------o
lopidSlid.
Day
2
Cow,
Day
3
Figure 3. Escherichia coli count of wounds as ascertained by rapid slide count and colony count.
tissue or greater, the number of organisms determined by the rapid slide technic did not differ significantly from that determined by the serial dilution and culturing technics. It is fortuitous that lo6 bacteria is the critical number of bacteria that will induce clinical infection in experimental animals and humans. The major advantage of the rapid slide technic is the speed with which the results are available to the surgeon. The culturing technic is obviously a valuable supplement to the rapid slide technic since it provides opportunity for speciation and antibiotic sensitivity testing.
761
Magee et al
When the level of bacteria in a wound biopsy was less than 2 X lo5 organisms per gram of tissue, bacteria usually were not visible on the slide. Failure to identify this low level of inoculum has little clinical significance since these small numbers of bacteria will not result in clinical infection. The rapid slide technic proved reliable for quantitation of gramnegative rods as well as the more easily recognized gram-positive cocci. The age of the wound at the time of biopsy did not alter the accuracy of the rapid slide technic. With serial dilution and culturing technics, eighteen of the thirty clinical specimens exhibited bacterial counts of less than 2 X lo5 organisms per gram of tissue; sixteen (89 per cent) were predicted by the rapid slide technic. Of the twelve samples containing more than 2 X lo5 bacteria, the number of bacteria in the specimen was accurately predicted to within half a logarithm of actual count obtained from nutrient agar plates.
basic changes, our accuracy in differentiating gram-positive from gram-negative organisms has improved considerably. The results of our rapid slide assay are reported as the number of bacteria per gram of tissue. Specifying the exact number of bacteria per gram of tissue allows the physicians to monitor any treatment effects. Demonstration of a reduction in wound bacterial count after initiating a treatment substantiates the benefits of that therapy. It is important to emphasize that the value of the rapid slide technic is realized primarily in the speed with which the results are available to the surgeon. The development of the rapid slide technic does not replace the quantitative serial dilution and plating technics. These latter technics are always performed concomitantly since they allow us to speciate the pathogen and perform antibiotic sensitivity testing. The results of these additional tests are essential to the management of the surgical patient with infection.
Comments
After repeated observations of the clinical significance of the number of bacteria present in tissue, a rapid method of performing quantitative wound cultures was developed by Heggers, Robson, and Doran [4]. Their technic was based on a direct microscopic examination of a measured aliquot of the homogenate. When we employed the technic in our laboratory, two problems were encountered. The technic was not reliable when the tissue sample size was smaller than 0.5 gm. When such minute specimens were studied, the total bacterial count susceptible to quantitation was considerably diminished. This problem was corrected by obtaining tissue samples weighing over 0.5 gm for rapid slide assay. We have established a useful guideline for physicians that allows them to estimate the weight of the specimen: 2 X 1 X 0.5 cm pieces of tissue weigh approximately 0.5 gm. The second major drawback in the rapid slide technic was our failure to differentiate bacteria accurately by the Gram stain technic. In many cases, gram-positive organisms were considered to be gram-negative. The technical factors contributing to the too easy decolorization of the gram-positive organisms were the method of fixation and the composition of the mordant. Heat fixation of the smear has been abandoned in favor of methanol fixation. The unstable mordant, Iz-KI, has been replaced by an iodophor with a long shelf life. With use of these
762
Summary
An improved technic has been developed to predict rapidly the critical number of bacteria in tissues. This technic gave reliable and accurate measurements for wounds containing more than 2 X lo5 organisms per gram of tissue. It is fortuitous that lo6 bacteria is the critical number of bacteria that will induce clinical infection in experimental animals and humans. The rapid slide technic is now being utilized routinely in our medical center to determine graft bed receptiveness, predict the safety of wound closure, and monitor the course of burns.
References 1. Krizek TJ, Robson MC, Kho E: Bacterial growth and skin graft survival. Surg Forum 18: 518, 1967. 2. Robson MC, Duke WF, Krizek TJ: Rapid bacterial screening in the treatment of civilian wounds. J Surg Res 14: 426, 1973. 3. Krizek TJ, Flagg SV, Wolfort FG, Jabaley ME: Delayed primary excision and skin grafting of the burned hand. Plest Recoflstr Surg 51: 524, 1973. 4. Heggers JP, Robson MC, Doran ET: Quantitative assessment of bacterial contamination of open wounds by a slide technique. Trans R Sot Trop Med Hyg 63: 532, 1969. 5. Magee CM, Rodeheaver G, Edgerton MT, Edlich RF: A more reliable Gram staining technic for diagnosis of surgical infections. Am JSurg 130: 341, 1975. 6. Madden J, Edlich RF, Schauerhamer R, Prusak M, Borner J, Wangensteen OH: Application of principles of fluid dynamics to surgical wound irrigation. Curr Top Surg Res 3: 85, 1971.
The American Journal of Surgery
