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Assessing Gram-stain error rates within the pharmaceutical microbiology

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Article in EJPPS EUROPEAN JOURNAL OF PARENTERAL AND PHARMACEUTICAL SCIENCES · April 2020
DOI: 10.37521/ejpps/25102

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EJPPS – European Journal of Parenteral and Pharmaceutical Sciences Volume 25 Issue 1
https://www.ejpps.online/
https://doi.org/10.37521/ejpps

Assessing Gram-stain error rates within the pharmaceutical microbiology laboratory

Tim Sandle Head of Microbiology, BPL, UK

Corresponding Author: Tim Sandle, Head of Microbiology

Bio Products Laboratory

England

Email: timsandle@btinternet.com
Abstract

Gram-staining remains the fundamental method for determinative bacteriology, dividing

bacteria into Gram-positive and Gram-negative organisms. This test provides information as
to the origin of any contamination and is a pre-requisite for many microbial identification
methods. Despite the longevity of the test, the test is highly reliant upon analyst technique
and therefore errors occur. While there are a few studies looking at errors in the clinical
context, research has not been extended to the pharmaceutical microbiology laboratory
context. In this study, we present a review of over 6,000 Gram-stains and establish an error
rate of around 3%, with the most common reason for error being an over-decolourisation
step resulting in organisms that should be Gram-positive appearing as Gram-negative. The
analysis enables others to benchmark their facilities against.

Introduction

Gram-staining, a differential staining technique, is of fundamental importance for the

identification and classification of bacteria and it is widely used in the medical and industrial
fields of microbiology. Despite being a long-established method, the technique remains open
to occasional errors. However, it is rare for the error rate to be quantified and assessed for
error types (with the few published studies being confined to the clinical setting) ¹,². Errors
related to issues with technique and interpretation. The interpretation of a Gram-stain slide
is inherently subjective. While interpretation can be overcome through good training there
remains several factors which can affect whether a correctly stained sample is obtained,
including specimen fixation, staining protocol, and slide analysis. In order to assess the level
of errors made in a typical laboratory, this paper assesses the different types of errors that
can occur within a pharmaceutical facility. Data was drawn from a pharmaceutical facility
located in south-east England.

The implications of errors with the Gram-stain can influence the selection of the test method
(and test kit) for the next stage of identification, whether this is a manual biochemical
identification method (such as API) or a semi-automated method (such as Vitek or Omnilog).
Getting an identification wrong could lead to an incorrect root cause analysis (which impacts
on all types of pharmaceutical processing, including sterile products) and potential errors
relating to batch release (especially with non-sterile pharmaceuticals where understanding
the pathogenic nature of the organism is a key requirement).

The Gram-stain

The Gram-stain is an important tool in the process of bacterial identification by dividing

bacteria into two groups (the so-called Gram-positives and Gram-negatives) and in allowing
their morphological types (coccoid or rod shaped) to be clearly seen ³. The developer of the
differential stain was the Danish bacteriologist Hans Christian Joachim Gram ⁴; it was during
his study of red corpuscles in 1884 that Gram developed his method for staining bacteria ⁵.

The polychromatic staining method has gone through several iterations since its initial
description ⁶. Today, the staining technique involves the following steps ⁷:
 • Taking a microbial colony and emulsifying it onto a slide.
• Fixing the smear to the slide through the application of heat.
• Staining bacterial cells with crystal violet.
• Fixing the stain (by using a mordant, chemicals that help “set” a dye, like iodine).
• Using a solvent to remove the stain from some types of bacteria, such as acetone or
ethanol.
• Applying a counter stain.

The idea for a counterstain was developed some years later by a German pathologist named
Carl Weigert who used safranin as a counter stain, which stained the cells red. Gram himself
never used a counter stain ⁸.

The first step involves taking single (pure) colonies from an agar plate of still growing bacteria
(often 18 – 24 hours old) and heat-fixing the cells (which kills them) onto a microscope slide.
Following this the cells are then stained with a basic dye, crystal violet, which stains all
bacterial cells blue. In aqueous solutions, crystal violet dissociates into CV+ and Cl – ions that
penetrate through the wall and membrane of both Gram-positive and Gram-negative cells.
The CV+ interacts with negatively charged components of bacterial cells, staining the cells
purple ⁹.

Figure 1: Positive Gram-stain (Micrococcus luteus) (Image: Tim Sandle)

The second step involves adding an iodine-potassium iodide solution. The iodine solution
enters the cells and forms a water-insoluble complex with the crystal violet dye. When added,
iodine (I- or I3-) interacts with CV+ to form large crystal violet-iodine (CV-I) complexes within
the cytoplasm and outer layers of the cell. For the third step, cells are treated with alcohol or
acetone solvent in which the iodine-crystal violet complex is soluble. Following solvent
treatment, only Gram-positive cells remain stained. The outer membrane of the Gram-
negative cell (lipopolysaccharide layer) is lost from the cell, leaving the peptidoglycan layer
exposed. Gram-negative cells have thin layers of peptidoglycan, one to three layers deep with
a slightly different structure than the peptidoglycan of gram-positive cells. With ethanol
treatment, Gram-negative cell walls is dissolved, and this allow the large CV-I complexes to
be washed from the cell, whereas the highly cross-linked and multi-layered peptidoglycan of
the Gram-positive cell is dehydrated by the addition of the solvent. The multi-layered nature
of the peptidoglycan along with the dehydration from the ethanol treatment traps the large
CV-I complexes within the cell ¹⁰.

After decolourisation, the Gram-positive cell remains purple in colour, whereas the gram-
negative cell loses the purple colour and is only revealed when the counterstain is added.
After the decolourisation procedure, cells are treated with a counterstain, i.e., a positively-
charged red acidic dye such as safranin, in order to make Gram-negative (decolourised) cells
visible (sometimes basic fuchsin is substantiated for safranin; fuchsin, generally, stains
bacteria more intensely than safranin. In addition, some bacteria are poorly stained by
safranin, such as Haemophilus spp., Legionella spp., and some anaerobic bacteria).
Counterstained Gram-negative cells appear red, and Gram-positive cells remain purple. The
slide is then examined microscopically using a x 1000 magnification (via a x10 eyepiece and a
x100 objective) under oil immersion (to improve the refractive index) ¹¹.

Figure 2: Negative Gram-stain (Bacteroides species) (Image: Tim Sandle)

Some bacteria cannot be stained using the Gram-method, these include bacteria that exist
almost exclusively within host cells such as intracellular bacteria (such as Chlamydia); those
that lack a cell wall (like the Mycoplasmas); and these of insufficient dimensions to be
resolved by light microscopy (such as the Spirochetes). In these cases, alternative stains can
be considered ¹².

Figure 3: Mixed Gram-stain (thermophilic Actinomycete) (Image: Tim Sandle)

Gram-stain errors

For an effective Gram-stain, the starting bacterial cell number needs to be sufficient (ideally
taken from a pure colony). To be visible on a slide, organisms that stain by the Gram method
must be present in concentrations of about 104 to 105 cells. Aside from this, errors can occur
with the Gram-stain and where errors lead to misidentifications, these can have serious
consequences.
The primary error is through mis-identifying Gram-positives as Gram-negatives and Gram-
negatives as Gram-positives. Of these two mis-categorizations the former is more common.
Gram-positive bacteria may lose their ability to retain crystal violet and stain Gram negatively
due to cell wall damage of bacteria due to antibiotic therapy or excessive heat fixation of the
smear or through the use of an Iodine solution which is too old, that is where the iodine turns
yellow instead of being brown in colour. However, the most common issue relates to over-
decolourisation of the slide smear. The key to the technique relates to the length of time that
the solvent is applied during the ‘decolourisation’ step, for too long an exposure removed the
stain from both groups of bacteria ¹³. A prolonged exposure to the decolourising agent will
remove all the stain from both types of bacteria. Some Gram-positive bacteria may lose the
stain easily and therefore appear as a mixture of Gram-positive and Gram-negative bacteria
(Gram-variable). Further, the phenomenon of ‘Gram-variability’ (or ‘Gram indeterminate’) is
also shown by ageing cells where logarithmic growth has been suspended, remains a problem.
What is happening is the thinning of the cell wall peptidoglycan layer, which occurs as some
cultures age. A further factor is where Gram staining imposes great stress on the integrity of
some organisms leading to a weakening of the protein S-layer ¹⁴.

It is also possible for Gram-negatives to appear as Gram-positives. This can occur when the
smear is too thick, resulting in Gram-negative bacteria not being fully decolourised during the
decolourisation steps and appearing as Gram-positive bacteria. Other errors include:

• Smear improperly heat-fixed.

• Iodine solution is weak.
• Failure to rinse between reagents.

Detection errors can also occur through the failure to use positive and negative control
smears on the slide, which means that procedural inaccuracies may go undetected.

Assessment of Gram-stain error rate

This paper presents a study of Gram-stain error rates across a two-year period, across
multiple laboratory analysts. The analysts worked at a major pharmaceutical facility located
in the south-east of England. Over 6,000 separate Gram-stain results were assessed for errors
and the errors were categorised, in an attempt to establish common causes for the errors.
With the study presented in this paper, errors were assessed by ¹⁵:
• Comparing the Gram-stain result to plate morphology
• Comparing the Gram-stain result to later species identification
• Assessing the Gram-stain by supervisory check and later repeat test

The types of errors were categorized into:

1. Misread stains (positive stains misread as negative and vice versa).
2. Mixed cultures.
3. Using subcultures that were aged (plated out >24 hours).
4. Over decolourisation.
5. Inadequate fixation (e.g. iodine solution is too weak).
6. Disorganisation resulting in slides being mislabelled and the incorrect result being
recorded for a given slide.
 7. Small specimen size.
8. Low number of bacterial cells.

Study results

During the course of the study, results from a total of 6,303 specimens (performed by ten
different analysts) were reviewed for discrepant smear Gram-stain results, as set out in Table
1. These tests were conducted over a two-year period (2016 to 2018). The incidence of
discrepant specimens was 216 (or 3.2% of the samples processed). The range across the
analysts was between 0% and 6.4%, with the mean error rate standing as 2.9%.

Table 1: Error rate by analyst

Analyst Number of Gram-stains No. (& percentage) of

prepared (n=6,303) discrepant results
1 1,030 36 (3.5%)
2 528 21 (3.9%)
3 374 14 (3.7%)
4 1,120 72 (6.4%)
5 905 33 (3.7%)
6 55 0 (0%)
7 327 11 (3.4%)
8 861 5 (0.6%)
9 1,005 22 (2.1%)
10 98 2 (2%)

In terms of the types of errors, Table 2 classifies the errors as Gram-stain results initially
recorded as Gram-negative (but where the bacterium was actually Gram-positive); where the
Gram-stain results initially recorded as Gram-positive (but where the bacterium was actually
Gram-negative); and where the result could not to be said to be conclusively either Gram-
negative or Gram-positive due to other factors (such as age of culture or due to a mixed
culture).
Table 2: Types of errors recorded by analyst according to incorrect Gram-stain outcome

Analyst No. (& percentage) No. (& percentage) No. (&

negative results that positive results that percentage)
should be positive should be negative indeterminant
stains
1 32 (3.1%) 4 (0.4%) 0 (0%)
2 15 (2.8%) 3 (0.6%) 3 (0.6%)
3 14 (3.7%) 0 (0%) 0 (0%)
4 60 (5.3%) 10 2
5 33 (3.7%) 0 (0%) 0 (0%)
6 0 (0%) 0 (0%) 0 (0%)
7 10 (3.1%) 0 (0%) 1 (0.3%)
8 4 (0.5%) 1 (0.6%) 0 (0%)
9 11 (1.1%) 6 (0.6%) 5 (0.5%)
10 2 (2%) 0 (0%) 0 (0%)
Total 181 24 11

With Table 2, the percentages are based on the number of tests performed by the analyst as
per Table 1. Table 2 shows that the most common error was with producing a Gram-negative
result in error when the bacterium was actually Gram-positive. This represented 181 of the
216 errors (83%). This type of error is consistent with over-decolourisation of the stain. The
second error, recording Gram-negative organisms erroneously as Gram-positive organisms
accounted for 24 incidents (of the 216 errors), 11% of the samples. In a few cases (11 of 204
or 5%) the result was indeterminant.

Table 3 provides a further breakdown of the reason for the errors, based on assessment by
supervisors and noting the performance of any repeat tests conducted.
Table 3: Detailed breakdown of error root causes, by number

Analyst Misread stains Mixed cultures Aged Over Inadequate Disorganisation Insufficient
subcultures decolourisatio fixation culture
n
1 1 2 1 32 0 0 0
2 1 3 2 14 0 1 0
3 0 0 0 12 1 0 1
4 2 4 2 60 0 4 0
5 1 1 0 31 0 0 0
6 0 0 0 0 0 0 0
7 2 0 0 9 0 0 0
8 2 1 0 2 0 0 0
9 1 5 4 11 0 0 0
10 0 0 0 2 0 0 0
Total 10 16 9 173 1 5 1
Based on the analysis presented in Table 3, the primary reason for errors relates to over-
decolourisation, at 173 of the 216 identified discrepant Gram-stains. This is followed by the
starting culture being mixed, at 16 and by misreading of stains (that is stains that were
correctly performed but which were misread by the analyst). The rank of errors is:

1. Over decolourisation
2. Mixed cultures
3. Misread stains
4. Aged subcultures
5. Disorganisation
6. Inadequate fixation and Insufficient culture

In terms of the types of organisms most likely to be susceptible to errors, this data was not
formally collated. However, the general pattern suggest that the over-decolourisation of
Bacillus species and related genera represented the largest bacterial group prone to Gram-
stain error. Where unchecked, this could lead to Bacillus species being mis-identified as Gram-
negative organisms (such as Pseudomonads). In terms of sample types, such
misidentifications tended to relate to environmental monitoring samples drawn from
cleanroom environments where medical products were prepared.

Discussion

Gram staining of bacterial cells is neither an accurate nor elaborate technique, but
nevertheless it is practically useful to distinguish two big domains of eubacteria species:
Gram-positive bacteria that include most of the Firmicutes, and Gram-negative bacteria that
include the remainder ¹⁶. Even in the emergence of more sophisticated microbial
identification methods, Gram-staining is required in many circumstances. Moreover, even
where manufacturers of microbial identification technologies state that Gram-staining is not
required (as with some molecular arrays) ¹⁷ or analysis of headspace volatile compounds ¹⁸,
should an atypical or discrepant result arise being able to refer back to the Gram-stain
invariably proves useful when conducting an investigation. Misidentification poses the risk of
sending any investigation down the wrong path which can cause further delays (and hence
impact on batch release) or present the person tasked with batch release (the Qualified
Person in Europe) with incorrect information and hence the risk of an incorrect decision being
made.

The data presented in this paper demonstrates that despite the long-history of the Gram-
stain, errors with the technique still occur, even with analysts who are well-trained and who
would be considered advanced practitioners.

In comparing the error rate to a pharmaceutical microbiology laboratory, the clinical error
rate was found to be 3.2% based on 6,303 specimens (based on discrepancy from culture) ¹⁹.
Notably there were some differences with the analysts in terms of their performance over
the two-year assessment period. The errors divided into specimens with no organisms
reported on Gram-stain but where significant growth on culture was observed; reported
organisms on a Gram-stain that were not recovered in culture; and discrepant results due to
reader error and so on, as set out in Tables 2 and 3. The primary reason for error was due to
over-decolourisation.

In terms of best practices designed to reduce error rate, this can be tackled by having clearly
defined standard operating procedures and putting analysts through robust training regimes.
Analyst competency takes on and even greater importance where the centralisation of
microbiology testing occurs, and activities become staffed by non-traditional microbiologists
²⁰. Training can be supported by participation in proficiency schemes ²¹. In addition, an
automated Gram-stainer may standardize the protocol; although such technology is not
without its limitations ²²,²³. Errors can also be reduced by using controls with each slide (one
organism designed to produce a positive reaction and another organism designed to produce
a negative reaction). Controls can either be prepared in the laboratory using known cultures
(such as those traceable to a national culture collection) or by using commercial slides with
the controls pre-prepared as dried-on smears. An additional practice to assess the difference
between Gram-positive and Gram-negative organisms is the potassium hydroxide test, which
assesses the difference in the cell wall, and this can supplement microscopic analysis ²⁴. Errors
can also be reduced through ensuring that the time steps for each stage of the staining
procedure are adhered to, such as by using a stopwatch.

The research presented has some limitations in that the error rates obtained using this
approach may only apply to the specific laboratory (rather than a multi-facility assessment)
and within one country. It may be that the actual error rate for Gram-stains in other
pharmaceutical microbiology laboratories may differ from the data presented here (as indeed
may laboratories with different functions, such as clinical laboratories). Furthermore, the data
presented represents a two-year window, which itself may or may not be truly representative.
Looking at the total data does not permit an evaluation as to whether the performance of
analysts improves over time (such as progressing from a trainee to a more experienced
analyst). However, the data presented in this paper is generally comparable to other studies
in the clinical setting. Samuel and colleagues recorded an error rate of around 5% across
different clinical facilities ¹⁹, which was a similar finding to Brizzi et al (also 5%) ²⁵; with Munson
and associates finding a 6% error rate ²¹. In different research, Rand and Tillan recorded a
lower error rate of around 1% ²⁶.

The data set out can go towards establishing a benchmark for the incidence of errors during
the performance of Gram-stains in the pharmaceutical and other disciplines and the types of
errors identified can be considered and addressed through laboratory training programmes,
with an aim to seek future improvements with the Gram-stain technique.
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