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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 4  |  Issue : 2  |  Page : 98-103

Comparison of four different methods of smear preparation for Gram staining of positively flagged automated blood culture bottles


Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, India

Date of Submission27-Sep-2018
Date of Acceptance10-Oct-2018
Date of Web Publication13-Dec-2018

Correspondence Address:
Apurba Sankar Sastry
Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry - 605 006
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrsm.jcrsm_34_18

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  Abstract 


Introduction: Gram staining of smears prepared from positively flagged blood culture bottles plays a vital role because its accurate interpretation helps the clinicians to modify empirical therapy as well as to perform the direct susceptibility test, which reduces the turnaround time by 24 h to initiate pathogen-directed antimicrobial therapy. However, the interpretation of direct Gram stain from blood culture bottles has always been at risk of errors in interpreting.
Aim: The present study was conducted to find the agreement between the blood culture broth Gram stain finding of four different methods (conventional, water wash, blood film, and drop and rest) of smear preparation with the culture smear Gram stain result and also to compare the quantity of charcoal and resin particles present in the Gram stained smears made from four different smear preparation methods.
Method and Result: Smears were made from 500 positively flagged automated blood culture bottles using the four different methods and then compared for agreement with the culture smear Gram stain using kappa statistics and were found to be maximum for the blood film method (0.637), followed by drop and rest (0.570), water-wash (0.496), and conventional method (0.475). The 3+ grading for resin/charcoal was found in 184 (36.80%) smears, 169 (33.80%), 93 (18.60%), and 26 (5.20%) smears prepared by water-wash, conventional, drop and rest, and blood film method, respectively. The interference caused by resin/charcoal was maximum when smears were prepared by the conventional method resulting in high inaccurate interpretation.
Conclusion: In this study, the agreement between direct smear preparation methods from positively flagged blood culture broth with the culture smear Gram stain was found to be maximum for the blood film method, followed by drop and rest, waterwash, and conventional method.It was also observed that the least amount of resin/charcoal was encountered when the direct smears were prepared using the blood film method.Hence, we found that the blood film method helps in interpreting the Gram stained smears from positively flagged blood culture broth more accurately compared to other methods.

Keywords: Blood culture, blood film method, conventional method, drop and rest method, Gram stain, water-wash method


How to cite this article:
Nain J, Deepashree R, Tamang P, Bhat P, Prakash S, Sneha R, Kalpana T, Anitha G, Sastry AS. Comparison of four different methods of smear preparation for Gram staining of positively flagged automated blood culture bottles. J Curr Res Sci Med 2018;4:98-103

How to cite this URL:
Nain J, Deepashree R, Tamang P, Bhat P, Prakash S, Sneha R, Kalpana T, Anitha G, Sastry AS. Comparison of four different methods of smear preparation for Gram staining of positively flagged automated blood culture bottles. J Curr Res Sci Med [serial online] 2018 [cited 2019 Mar 19];4:98-103. Available from: http://www.jcrsmed.org/text.asp?2018/4/2/98/247496




  Introduction Top


The Gram stain although one of the oldest techniques used in the microbiology laboratory still remains the cornerstone of all the diagnostic tests for the guidance of empirical antibiotics treatment before the availability of bacterial culture results.[1] The role of Gram staining is vital in blood culture investigations, as most patients for whom blood cultures are sent are critically ill. Accurate Gram stain results from positively flagged bottles not only help the clinicians to modify empirical therapy but also help in performing the direct susceptibility test, which reduces the turnaround time by 24 h to initiate pathogen-directed antimicrobial therapy.[1],[2] Studies have shown that inaccurate interpretation of Gram stain of positively flagged blood culture bottles has a direct impact in terms of poor clinical outcome.[1],[2],[3],[4]

The interpretation of direct Gram stain from blood culture bottles is not an easy task and has always been at risk of errors in interpreting. Several factors contribute to this misinterpretation such as technical skill of the observer, variable level of bacteremia in patients, lower bacterial load in liquid culture compared to colony, and presence of resins and charcoal in blood culture medium. Of these, the resins and charcoal particles present in the medium appear to be the most important confounding factor.[2],[3],[5],[6] Although resin and charcoal help in absorbing antimicrobials and thus are associated with higher yield rate, several studies have reported that they interfere with the reading of Gram stained smears.[5]

Resins and charcoal residues present in the broth are stained Gram-positive, appear either scattered or clumps of various sizes, and interfere with the interpretation.[5] While through practice and familiarity, observers get used to and can make out the differences, this is not the case always. An appropriately made smear that reduces the amount of resin and charcoal residues on the slide will aid in the accurate interpretation of Gram stained smears.

The conventional smear method that is commonly used for any liquid culture has been associated with a higher inaccurate result when performed for blood culture broth.[3] There are several other methods proposed in the literature such as water washing method, blood film method, and drop and rest method. The latter two methods have been recommended by the manufacturer of automated blood culture bottles.[7] The blood film method evenly distributes the sample, separating the charcoal residue into a thin thumbprint smear containing microorganisms. The drop and rest method also makes the smear free from resins and charcoals and helps in making the interpretation easy.

To the best of our knowledge, there is no study available on assessing effects on smear preparation methods from blood culture broth on the interpretation of Gram stained smear. Therefore, this study was undertaken to find the agreement between blood culture broth Gram stain findings of four different methods (conventional, water washing, thin smear, and drop and rest) of smear preparation with the culture smear Gram stain result.


  Methodology Top


It was a prospective cohort study, conducted at Jawaharlal Institute of Medical Education and Research Institute, located at Puducherry, India, during February 2018 to August 2018. Positively flagged automated bottles of two types, Bact/Alert FA plus and BACTEC Plus Aerobic, were included in the study. The positively flagged bottles were removed from the instrument(s), taken to biological safety cabinet, and then, checked for sign of increased gas pressure such as bulging septum and overfilling which were relieved by allowing the bottle to come to room temperature. When the positive bottles were out of the instrument for 15 min or more, the broth was resuspended by gently inverting bottle several times before use. The bottle septum was cleaned with an individual sterile alcohol pad which was left on the bottle top. The needle of a sterile unused syringe was then inserted through the alcohol pad, and 1–2 ml of broth was aspirated. Four different types of smear preparation methods (conventional method, blood film method, drop and rest method, and water-wash method) were carried out according to the standard reference as described below.[7],[8],[9],[10],[11]

Conventional method

A drop of sample was placed onto a clean slide and then spread to make a smear of 1 cm diameter. The smear was dried and then heat fixed.[8]

Water-wash method

A drop of blood from the culture bottle was placed onto a clear and clean labeled slide and then dried. Then, the slide was washed gently with water allowing the tap water to fall on thumb first then free flowing on the smear. Then, the smear was heat fixed and stained.

Blood film method

This method is similar to making a thin peripheral blood smear. Procedure was followed according to the manufacturer's recommendation.[7] After transferring a small drop of sample onto the end of clean labeled slide, another clean slide was placed at 25° angle over the first slide to hold the drop of the blood. Then, the spreader slide was firmly pressed against the first slide and then pushed forward smoothly and rapidly to spread the sample thinly over the entire slide. Then, the slide was dried, heat fixed, and stained. The stained smear was read in the slide area where the sample was thinly spread.

Drop and rest method

Drop and rest method is another recommended method for making smears of organisms recovered in blood culture bottles.[7] A drop of sample was placed onto a clean labeled slide and then allowed to rest for 10 min, allowing the charcoal particles in the sample to settle. The slide was tilted so that a stream of the sample was separated from the charcoal and flown onto the slide. Then, the smear was spread using an applicator stick. The slide was air dried and heat fixed at 60°C or fixed with 95% methanol for 2 min. After staining, the slide was read in the area where the sample was spread out, not the area where the sample drop was placed.

The smears prepared by all the above-mentioned methods were subjected to Gram stain according to the standard method.[11] As part of the routine processing of microbiology laboratory, the broth from positively flagged blood culture bottles was streaked on routine bacteriological media such as sheep blood agar and MacConkey agar. Gram staining of the culture smears was done from the colonies grown after overnight incubation at aerobic 37°C.

To have uniformity in reporting, (i) the Gram stain interpretations were categorized into five variables: Gram-positive cocci, Gram-positive bacilli, Gram-negative cocci, Gram-negative bacilli, and mixture, and (ii) the amount of resins or charcoals present in the smear was expressed as nil (if no resins/charcoals observed in 20 fields), minimum or + (if resin/charcoals observed in 1–5 of 20 fields), moderate or ++ (if resin/charcoals observed in 6–10 fields of 20), and heavy or +++ (resins/charcoals observed in >10 of 20 fields).

The smear preparation and Gram staining were performed by the same trained laboratory technician throughout the study. The slides were read by three independent trained observers who had at least 3 years of experience in interpreting Gram stain results. The observers were kept blinded about the culture smear results until the end of the study. The reading was considered as final interpretation only when at least two out of three observers agreed on the interpretation. Those smears where all three observers interpreted differently were not considered for analysis.

Parameters Studied

The agreement between the blood culture broth Gram stain finding of all four smear preparation methods with the culture smear Gram stain result was found out. The quantity of charcoal and resin particles present in the Gram stained smears made from four smear preparation methods were also compared.

Statistical tests used for data analysis

The agreement between the blood culture broth Gram stain finding of all four smear preparation methods with the culture smear Gram stain result was statistically analyzed using kappa statistics.

Ethical considerations

Ethical clearance was obtained from the Institute Ethics Committee, and waiver of consent was obtained as data were collected from the laboratory without any direct or indirect patient involvement.


  Results Top


Smears were made from 500 positively flagged automated blood culture bottles using the four different methods and then compared for agreement with the culture smear Gram stain. The agreement between direct smear preparation methods from positively flagged blood culture broth with the culture smear Gram stain was statistically analyzed using kappa statistics and was found to be maximum for the blood film method (0.637), followed by drop and rest (0.570), water wash (0.496), and conventional method (0.475) [Table 1] and [Figure 1].
Table 1: Agreement between different direct smear preparation methods from positively flagged blood culture broth with the culture smear Gram stain

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Figure 1: Agreement between different direct smear preparation methods from positively flagged blood culture broth with the culture smear Gram stain

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[Table 2] and [Figure 2] illustrate that Gram-positive cocci in clusters were better interpreted by the blood film (155/202, 76.70%), conventional (153/202, 75.70%), and drop and rest methods (152/202, 75.20%), whereas the water-wash method could correctly interpret only 59.4% (120/202) of GPC clusters. Gram-positive cocci in chains were more accurately interpreted by the blood film method (5/7, 71.40%) followed by the conventional (3/7, 42.90%), drop and rest (3/7, 42.80%), and water-wash (3/7, 42.80%) method. Gram-positive cocci in pairs were better interpreted by the blood film (9/13, 69.20%) and drop and rest method (9/13, 69.20%), followed by the water wash (6/13, 46.10%) and the conventional methods (4/13, 30.80%). About 91.6% (11/12) of budding yeast cells were accurately interpreted by both blood film and drop and rest method followed by the conventional (10/12, 83.30%) and water-wash method (9/12, 75%). Nearly 79.20% of Gram-positive rods were correctly interpreted by the blood film (42/53) method followed by water wash (26/53, 49%), drop and rest (25/53, 47.10%), and conventional method (14/53, 26.40%).
Table 2: Comparison of different methods based on final identification

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Figure 2: Grading of resin/charcoal

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Gram-negative rods were better interpreted by blood film method (119/152, 78.20%), followed by drop and rest (111/152, 73%), conventional (110/152, 72.40%), and water-wash method (90/152, 59%). About 50% of slides prepared by drop and rest method (30/60) which were read as “no bacteria seen” (NBS) was also found to be culture sterile, followed by water wash (28/60, 46.60%) and blood film method (26/60, 43.30%). Only 20% of smears prepared by the conventional method (12/60) which were read as NBS was actually found to be sterile [Table 2] and [Figure 2].

[Table 3] shows that, of 500 Gram stained smears prepared by four different methods, resin/charcoal deposits were found to be heavy (3+) in 184 (36.80%) smears and in 169 (33.80%) smears prepared by water wash and conventional method, respectively. The 3+ grading was found in 93 (18.60%) smears prepared by drop and rest method and in only 26 (5.20%) smears prepared by the blood film method. Two hundred smears (40%) prepared by conventional method, 190 smears (38%) by drop and rest, 165 smears (33%) by water wash, and 101 smears (20.20%) by blood film method were found to have moderate (2+) quantity of resin/charcoal. The 1+ grading of resin/charcoal was found in 238 (47.60%) smears prepared by blood film method, 176 smears by drop and rest (35.20%), 124 smears by water wash (24.80%), and 102 smears by conventional method (20.40%). Resin/charcoal deposits were observed to be nil in 135 smears (27%) prepared by the blood film method and in 41 smears (8.20%) prepared by drop and rest method, whereas only 29 smears (5.80%) and 27 smears (5.4%) prepared by the conventional and water-wash method, respectively, were observed to have nil resin/charcoal.
Table 3: Grading of resin/charcoal

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  Discussion Top


Bloodstream infection (BSI) is a serious condition with an overall mortality above 20%. Early administration of appropriate empirical antibiotic treatment is known to be associated with better patient outcome. It is observed that up to 40% of all patients with BSI receive inappropriate antimicrobial treatment until the first notification of a positive blood culture. Therefore, an imperative for the diagnostic microbiology laboratory is to provide accelerated reports on positive blood cultures to guide antimicrobial therapy. It is also noted that stepwise intimation of blood culture report at different stages such as direct Gram stain, culture identification, direct antimicrobial susceptibility testing, and final report with antimicrobial susceptibility testing has a greater impact on guiding antimicrobial therapy rather communicating only the final report to the clinicians.[12],[13]

Among all the stages of reporting, Gram stain is the simplest and most rapid test to detect organisms directly from positive blood culture bottles, and therefore, it helps in instituting adequate antimicrobial agents as soon as possible. The American Society for Microbiology indicates that the single most important first test to be performed on any positive blood culture is Gram stain and states that the terminology used to report such results should be as descriptive as possible. The first notification of a positive blood culture is typically based on the Gram stain result.[12],[13]

Accurate interpretation of Gram stain smears from positively flagged blood culture broth helps the clinicians to modify empirical therapy and helps the laboratory personnel to perform the direct susceptibility test, thereby reducing the turnaround time by 24 h to initiate pathogen-directed antimicrobial therapy.[1],[2] Various studies have shown that notifying the Gram stain report results in initiation of antimicrobial therapy in 12%–20% of the patients and change to appropriate empirical antimicrobial treatment in another 30%–45% of patients.[12],[13] However, inaccurate Gram stain report may lead to delay in the initiation of appropriate antimicrobials.

The erroneous Gram stain report may be due to various reasons such as poor smear preparation technique, the presence of resins and/or charcoal particles of broth in the background smear interfering with Gram stain interpretation, and an inexperienced microscopist. Therefore, it is important for the microbiology laboratory to develop a better smear preparation technique which can yield improved interpretation. Studies have reported that the conventional smear method has been associated with a higher inaccurate result.[3] Apart from the conventional method, there are other methods also such as water wash, drop and rest, and blood film methods for preparing smears from blood culture broth. Keeping this in mind, this study has been undertaken to compare various smear preparation techniques to standardize an improved method which can be implemented in the future as routine.

In this study, the blood film method followed by drop and rest method was found to be associated with a higher agreement with the culture smear Gram stain result compared to other methods. It was also found that the interference caused by the presence of resin/charcoal was least when prepared using the blood film method making the interpretation of Gram stained smears of blood culture broth more easy and accurate. The resin and charcoal residues interfered the most when smears were prepared using the conventional and water-wash methods, making the interpretation difficult and inaccurate.

Blood film method was found to be superior for detection of Gram-positive cocci in cluster (76.7%), Gram-positive cocci in chain (71.4%), Gram-positive rods (79.2%), Gram-negative bacilli (78.2%), and budding yeast cells (91.6%). Both blood film and drop and rest methods were equally superior for detection of Gram-positive cocci in pairs (69.2%). These findings support the use of blood film method as part of routine direct Gram staining from positively flagged blood culture broth.

A study conducted by Søgaard et al. documented the sensitivity of direct Gram staining from blood culture broth to be 95.5% for Gram-positive cocci in clusters, 94.8% for Gram-positive cocci in chains/diplococci, 89.3% Gram-positive rods, 96.6% for Gram-negative rods, and 95.7% for yeasts. The higher sensitivity in this study could be due to training provided to the technical team for performing Gram staining. More so, this study did not differentiate between various smear preparation methods.[13]

The conventional method was found to be associated with a higher number of false-positive interpretation which could possibly be due to the presence of higher amount of resins and charcoal residues. The drop and rest method was found to give the least number of false-positive interpretations, followed by the blood film and water-wash method. The water-wash method is more time consuming compared to other methods, and there is a risk of washing off the smear during the preparation.

To the best of our knowledge, this study is the first of its kind, comparing the four different methods of smear preparation from blood culture broth based on agreement with the culture smear Gram stain.


  Conclusion Top


The resins and charcoal particles present in the blood culture medium are one of the several factors interfering with the reading of direct Gram stained smears resulting in inaccurate interpretation. In this study, the agreement between direct smear preparation methods from positively flagged blood culture broth with the culture smear Gram stain was found to be maximum for the blood film method, followed by drop and rest, water-wash, and conventional method. It was also observed that the least amount of resin/charcoal was encountered when the direct smears were prepared using the blood film method, followed by drop and rest and water-wash method. The interference caused by resin/charcoal was maximum when smears were prepared by the conventional method resulting in high inaccurate interpretation. Hence, we found that the blood film method helps in interpreting the Gram stained smears from positively flagged blood culture broth more accurately, as compared to other methods (water-wash, drop and rest, and conventional method).

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Samuel LP, Balada-Llasat JM, Harrington A, Cavagnolo R. Multicenter assessment of gram stain error rates. J Clin Microbiol 2016;54:1442-7.  Back to cited text no. 1
    
2.
Rand KH, Tillan M. Errors in interpretation of gram stains from positive blood cultures. Am J Clin Pathol 2006;126:686-90.  Back to cited text no. 2
    
3.
Behera B, Mathur P, Gupta B. Blood culture gram stain, acridine orange stain and direct sensitivity-based antimicrobial therapy of bloodstream infection in patients with trauma. Indian J Med Microbiol 2010;28:138-42.  Back to cited text no. 3
[PUBMED]  [Full text]  
4.
Adler H, Baumlin N, Frei R. Evaluation of acridine orange staining as a replacement of subcultures for bacT/ALERT-positive, gram stain-negative blood cultures. J Clin Microbiol 2003;41:5238-9.  Back to cited text no. 4
    
5.
Fiori B, D'Inzeo T, Di Florio V, De Maio F, De Angelis G, Giaquinto A, et al. Performance of two resin-containing blood culture media in detection of bloodstream infections and in direct matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) broth assays for isolate identification: Clinical comparison of the BacT/Alert plus and bactec plus systems. J Clin Microbiol 2014;52:3558-67.  Back to cited text no. 5
    
6.
Kristóf K, Pongrácz J. Interpretation of blood microbiology results – Function of the clinical microbiologist. EJIFCC 2016;27:147-55.  Back to cited text no. 6
    
7.
BACT/ALERT® FAN® PLUS Gram Stain; 2018. Available from: https://www.biomerieuxuniversity.com/bact-alert-fan-plus-gram-stain.html. [Last accessed on 2018 Feb 15].  Back to cited text no. 7
    
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Tille P. Bailey & Scott's Diagnostic Microbiology. 13rd ed. St. Louis, Missouri: Elsevier Health - US; 2013.  Back to cited text no. 8
    
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Available from: http://www.bmsd.com.my/files/editor_files/file/2_-February-2013.pdf. [Last accessed on 2017 Oct 26].  Back to cited text no. 9
    
10.
Winn WC, Koneman EW. Koneman's Color Atlas and Textbook of Diagnostic Microbiology. 6th ed. Philadelphia: Lippincott Williams & Wilkins; 2006.  Back to cited text no. 10
    
11.
Mackie TJ, McCartney JE. Mackie & McCartney Practical Medical Microbiology. 14th ed. New York: Churchill Livingstone; 1996.  Back to cited text no. 11
    
12.
Uehara Y, Yagoshi M, Tanimichi Y, Yamada H, Shimoguchi K, Yamamoto S, et al. Impact of reporting gram stain results from blood culture bottles on the selection of antimicrobial agents. Am J Clin Pathol 2009;132:18-25.  Back to cited text no. 12
    
13.
Søgaard M, Nørgaard M, Schønheyder HC. First notification of positive blood cultures and the high accuracy of the gram stain report. J Clin Microbiol 2007;45:1113-7.  Back to cited text no. 13
    


    Figures

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    Tables

  [Table 1], [Table 2], [Table 3]



 

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