|Year : 2014 | Volume
| Issue : 4 | Page : 168-174
Laboratory perspective of gram staining and its significance in investigations of infectious diseases
Yunusa Thairu1, Idris Abdullahi Nasir1, Yahaya Usman2
1 Department of Medical Microbiology, University of Abuja Teaching Hospital, Gwagwalada, Abuja, Nigeria
2 Department of Medical Laboratory Science, Faculty of Medicine, Ahmadu Bello University, Zaria, Kaduna, Nigeria
|Date of Submission||03-Jun-2014|
|Date of Acceptance||11-Sep-2014|
|Date of Web Publication||14-Nov-2014|
Department of Medical Microbiology, University of Abuja Teaching Hospital, P.M.B. 228, Gwagwalada, Abuja
Clinical microbiology laboratory plays several important roles in the management of bacterial infections. Isolation, identification of pathogenic microorganisms in cultures and subsequent antimicrobial susceptibility testing always assists in selecting appropriate antimicrobial agent and prevention of unnecessary complications. The most important and primary test to perform directly on some special samples such as cerebrospinal fluid and positive cultures is Gram staining which serves as the most rapid and simplest test to characterize microorganisms. It is therefore highly likely that the information provided by the Gram staining will help to assess the adequacy of preliminary diagnosis and antimicrobial therapy selected after collecting culture specimens and before final identification of the microorganism. In recent reports, the impact of Gram staining results on patient mortality has been documented. On the other hand, there remains the possibility that Gram staining results do not match with the final identification of microorganisms. This would carry a risk leading to inadequate antimicrobial therapy and potentially affecting patients' clinical course and mortality. The aim of this mini review is to analyze and discuss the clinical significance and limitations of reporting Gram staining results for sample meant for bacteriological investigations.
Keywords: Positive culture, Bacteriological, Limitations and special samples
|How to cite this article:|
Thairu Y, Nasir IA, Usman Y. Laboratory perspective of gram staining and its significance in investigations of infectious diseases. Sub-Saharan Afr J Med 2014;1:168-74
|How to cite this URL:|
Thairu Y, Nasir IA, Usman Y. Laboratory perspective of gram staining and its significance in investigations of infectious diseases. Sub-Saharan Afr J Med [serial online] 2014 [cited 2019 Jan 18];1:168-74. Available from: http://www.ssajm.org/text.asp?2014/1/4/168/144725
| Introduction|| |
Effective utilization and understanding of the clinical bacteriology laboratory can greatly aid in the diagnosis of infectious diseases. , Although described more than a century ago, the Gram stain remains the most frequently used rapid diagnostic test, and in conjunction with various biochemical tests is the cornerstone of the clinical laboratory. First described by Danish pathologist Christian Gram in 1884 and later slightly modified. ,
The Gram stain easily divides bacteria into two groups, Gram-positive and Gram-negative, on the basis of their cell wall and cell membrane permeability. The mechanism further implies that solvent decolorization causes significant damage to the cell surfaces of Gram-negative bacteria and only limited damage to Gram-positive bacteria. This suggests Gram-negative bacteria are more "leaky," causing these thin-walled lipid-rich cells to lose their crystal violet (CV) stain and appear red from the counterstain. Gram-positive cells, thick walled and lipid-poor, appear blue from retaining the original CV.  The Gram stain is not an infallible tool for diagnosis, identification, or phylogeny, however. It is of extremely limited use in some instances and has been largely superseded by molecular techniques even in the medical microbiology laboratory. Given that some organisms are Gram-variable (i.e., they may stain either negative or positive), and that some organisms are not susceptible to either stain used by the Gram technique, its true utility to researchers should be considered limited and specific. In molecular microbiology laboratory, most identification is done using genetic sequences and other molecular techniques which are far more specific and information-rich than differential staining. 
The Gram stain has been used for several purposes that include:
- To directly examine specimens submitted for microbiologic examination, e.g., body fluid or biopsy when infection is suspected. It yields results much more quickly than culture, and is especially important when infection would make an important difference in the patient's treatment and prognosis; examples are cerebrospinal fluid for meningitis and synovial fluid for septic arthritis
- To provide preliminary information to the clinician regarding presumptive bacterial pathogens
- To characterize the type of bacteria growing in culture media (including blood cultures)
- And to assess the quality of specimens submitted for culture (i.e., sputum specimens) to determine whether they are likely to yield clinically useful information and whether they should be processed (cultured).
This specimen quality assessment is possible because the leukocytes and epithelial cells of the human host also stain with the Gram stain. 
As a general rule of thumb (which has exceptions), Gram-negative bacteria are more pathogenic due to their outer membrane structure. The presence of a capsule will often increase the virulence of a pathogen. In addition, Gram-negative bacteria have lipopolysaccharide (LPS) in their outer membrane, an endotoxin that increases the severity of inflammation. This inflammation may be so severe that septic shock may occur. Gram-positive infections are generally less severe because the human body does not contain peptidoglycan; in fact, humans produce an enzyme (lysozyme) that attacks the open peptidoglycan layer of Gram-positive bacteria. Gram-positive bacteria are also frequently much more susceptible to beta-lactam antibiotics, such as penicillin. 
Exceptions to the rule include branching and filamentous Gram-positive bacteria such as Mycobacterium tuberculosis and other agents of tuberculosis, or Nocardia species, the agents of nocardiosis and some types of actinomycetoma.  These organisms present unique problems in diagnosis and treatment, and special stains such as the Ziehl-Neelsen stain and the Kinyoun stain are used in their laboratory workup.
Like other laboratory tests, the Gram stain has certain inherent limitations and is subject to technical variation and misinterpretation - the subject of an article by Rand and Tillan.  This retrospective study reviews major errors, defined by the authors as errors in which the original Gram stain of a positive blood culture reports a single organism whose Gram stain morphology is opposite (Gram-positive vs. Gram-negative or vice versa) to the Gram stain morphology of the final culture organism identification. The authors found that in 57 (0.7%) of 8,253 patients with positive blood cultures the Gram stain was misread, resulting in major errors that fell into three categories:
- Six cases where the original Gram stain report was Gram-positive cocci and the culture yielded Gram-negative bacilli, 3 of which were Acinetobacter species.
- Twenty-five cases where the original Gram stain report was Gram-negative bacilli and the culture yielded Gram-positive organisms; of these were Bacillus species and 2 were Clostridium species (this type error is probably due to overdecolorization, a common technical problem in Gram stains).
Twenty-eight cases where the original Gram stain showed a single organism but the culture yielded multiple organisms; most often in this group the original Gram stain showed Gram-positive cocci. When the medical charts of these patients were reviewed, there were four patients in whom delays of 14 h to 3 days occurred in starting appropriate antibiotics, with two deaths, although the authors believed that the erroneous Gram stain report was probably not contributory.
Although the study by Rand and Tillan has limitations (e.g., the reasons for the errors in the Gram stain reports - technical artifact, interpretation error, or clerical error - could not be specifically determined in this retrospective study), it is still a useful contribution to the field of clinical microbiology for several reasons. First, the data presented show that the overall performance of the clinical laboratory scientists in interpreting Gram stains (99.31% of the Gram stains were interpreted accurately over 23 months was very good, especially when one considers that the Gram stain morphology of bacteria can be affected by antibiotic therapy, technical variables including overdecolorization and underdecolorization, and misinterpretation by those reading the Gram stains. Although 100% accuracy in reporting is always the goal of the laboratory, the accuracy of >99% for reporting of positive blood cultures, which are critical laboratory values, is to be commended.
Second, the authors provide important information to laboratory directors by identifying the most common types of major errors in blood culture Gram stains so that the directors can emphasize these problems in training and continuing education of the bench microbiologists responsible for interpreting Gram stains. Third, the finding that a frequent error was reporting a single organism when 2 or more were present prompts microbiologists to continue to vigorously search for other organisms, even after identifying 1 organism in a blood culture Gram stain, especially since Gram-positive organisms are generally more easily visualized than Gram-negative organisms.
Clinical Significance of Gram Staining
The Gram stain is a very important preliminary step in the initial characterization and classification of bacteria. It is also a key procedure in the identification of bacteria based on staining characteristics, enabling the bacteria to be examined using a light microscope. The bacteria present in an unstained smear is invisible when viewed using a light microscope. Once stained, the morphology and arrangement of the bacteria may be observed as well. Furthermore, it is also an important step in the screening of infectious agents in clinical specimens such as direct smears from a patient. Although the Gram staining is used for detection and differentiation of bacteria, other microorganisms, most frequently yeasts and fungi, can be seen on a Gram-stained smear.  Like Clostridium organisms, yeasts can appear Gram-positive or Gram-negative. Yeasts are generally at least 10-20 times the size of bacteria and hence differentiation from bacteria is not a problem. However, yeasts are the size of CV precipitate, which is occasionally present.
This large purple structure can even appear to be budding. CV precipitate can be differentiated from yeasts because:
- The precipitate may be present in an area with several other purple artifacts of various size and shape,
- The precipitate has a homogenous deep purple color, while the yeast is often mottled, and
- The precipitate may be perfectly round, while Candida species, the yeast most commonly encountered, are generally oval. 
Below are list of commonly encounter pathogens on Gram stain smears.
Stain dark purple due to retaining the primary dye called CV in the cell wall. Example Gram positive stained colonies of Candida spps showing typical large oval budding cells and extensive pseudohypae [Figure 1]a and 1b respectively]; Gram stained colonies of Staphylococus aureus recovered from Blood culture of a baby with sepsis [Figure 2].
|Figure 2: Gram stained colonies of Staphylococus aureus recovered from Blood culture of a baby with sepsis, source: www.cdc.org/ncidod/diseases/submenu/sub_staphylococcus|
Click here to view
|Figure 4: Gram negative Coccobacilli of Haemophilus infl eunzae recovered from a fi ve-year old child with cerebrospinal meningitis, source: www.tommytoy.typepad.com|
Click here to view
Stain red or pink due to retaining the counter staining dye called Safranin or neutral red. Example: Gram Negative Bacilli of Escherichia More Details coli from a woman suffering from Urinary tract infections [Figure 3] and Gram negative Coccobacilli of Haemophilus infleunzae recovered from a five-year old child with cerebrospinal meningitis [Figure 4].
|Figure 3: Gram Negative Bacilli of Escherichia coli from a woman suffering from Urinary tract infections, www.cdc.org/ecoli|
Click here to view
| Gram stain mechanism|| |
Gram-Positive Cell Wall
Gram-positive bacteria have a thick mesh-like cell wall which is made up of peptidoglycan (50-90% of cell wall), which stains purple. Peptidoglycan is mainly a polysaccharide composed of two subunits called N-acetyl glucosamine and N-acetyl muramic acid. As adjacent layers of peptidoglycan are formed, they are cross-linked by short chains of peptides by means of a transpeptidase enzyme, resulting in the shape and rigidity of the cell wall. The thick peptidoglycan layer of Gram-positive organisms allows these organisms to retain the CV-iodine complex and stains the cells as purple. 
Lipoteichoic acid is another major constituent of the cell wall of Gram-positive bacteria that is embedded in the peptidoglycan layer. It consists of teichoic acids that are long chains of ribitol phosphate anchored to the lipid bilayer through a glyceride. It acts as a regulator of autolytic wall enzymes (muramidases: Bacterial enzymes located in the cell wall that causes disintegration of the cell following injury or death).
Lipoteichoic acid also has antigenic properties that stimulate specific immune responses when it is released from the cell wall after cell death. Cell death is mainly due to lysis induced by lysozymal activities, cationic peptides from leucocytes, or beta-lactam antibiotics. 
Gram-Negative Cell Wall
Gram-negative bacteria have a thinner layer of peptidoglycan (10% of the cell wall) and lose the CV-iodine complex during decolorization with the alcohol rinse, but retain the counter stain Safranin, thus appearing reddish or pink. They also have an additional outer membrane, which contains lipids, which is separated from the cell wall by means of the periplasmic space. 
Relevance of Gram-Negative Cell Wall
The cell wall of Gram-negative bacteria is often a virulence factor that enables pathogenic bacteria to cause disease. The virulence of Gram-negative bacteria is often associated with certain components of the cell wall, in particular, the LPS (otherwise known as LPS or endotoxin). In humans, LPS elicits an innate immune response characterized by cytokine production and activation of the immune system. Inflammation occurs as a result of cytokine production, which can also produce host toxicity. 
| Stain reaction|| |
The four basic steps of the Gram stain (as described by WHO, 2013) are. 
Application of the Primary Stain to a Heat-Fixed Smear of Bacterial Culture
CV dissociates in aqueous solutions into CV+ and Cl- ions. These two ions then penetrate through the cell wall and cell membrane of both Gram-positive and Gram-negative cells. The CV + ions later interact with negatively charged bacterial components and stains the bacterial cells purple.
Addition of Gram's Iodine
Iodine (I− or I3−) acts as a mordant and as a trapping agent. A mordant is a substance that increases the affinity of the cell wall for a stain by binding to the primary stain, thus forming an insoluble complex, which gets trapped in the cell wall. In the Gram stain reaction, the CV and iodine form an insoluble complex (CV-I), which serves to turn the smear a dark purple color. At this stage, all cells will turn purple.
Decolorization with 95% Ethyl Alcohol
Alcohol or acetone dissolves the lipid outer membrane of Gram-negative bacteria, thus leaving the peptidoglycan layer exposed and increases the porosity of the cell wall. The CV-I complex is then washed away from the thin peptidoglycan layer, leaving Gram-negative bacteria colorless.
On the other hand, alcohol has a dehydrating effect on the cell walls of Gram-positive bacteria that causes the pores of the cell wall to shrink. The CV-I complex gets tightly bound into the multi-layered, highly cross-linked Gram-positive cell wall thus staining the cells purple.
The decolorization step must be performed carefully. Otherwise over-decolorization may occur. This step is critical and must be timed correctly otherwise the CV stain will be removed from the Gram-positive cells. If the decolorizing agent is applied on the cell for too long time, the Gram-positive organisms to appear Gram-negative. Under-decolorization occurs when the alcohol is not left on long enough to wash out the CV-I complex from the Gram-negative cells, resulting in Gram-negative bacteria to appear Gram-positive.
Counterstain with Safranin
The decolorized Gram-negative cells can be rendered visible with a suitable counterstain, which is usually positively charged safranin, which stains them pink. Pink color that adheres to the Gram-positive bacteria is masked by the purple of the CV (basic fuschin is sometimes used instead of safranin in rare situations).
It is a prudent practice to always include a positive and negative control on the staining procedure to confirm the accuracy of the results  and to perform proficiency testing on the ability of the Laboratory scientist to correctly interpret the stains. 
| Errors during gram staining|| |
It is clear that the decolorization step is the one most likely to cause problems in the Gram stain. The particular concerns in this step are listed below. 
Excessive heat during fixation
Heat fixing the cells, when done to excess, alters the cell morphology and makes the cells more easily decolorized.
Low concentration of crystal violet
Concentrations of CV up to 2% can be used successfully, however low concentrations result in stained cells that are easily decolorized. The standard 0.3% solution is good, if decolorization does not generally exceed 10 s.
Excessive washing between steps
The CV stain is susceptible to wash-out with water (but not the CV-iodine complex). Do not use more than a 5 s water rinse at any stage of the procedure.
Insufficient iodine exposure
The amount of the mordant available is important to the formation of the CV-iodine complex. The lower the concentration, the easier to decolorize (0.33-1% commonly used). Furthermore, QC of the reagent is important as exposure to air and elevated temperatures hasten the loss of Gram's iodine from solution. A closed bottle (0.33% starting concentration) at room temperature will lose >50% of available iodine in 30 days, an open bottle >90%. Loss of 60% iodine results in erratic results.
95% ethanol decolorizes more slowly, and may be recommended for inexperienced technicians while experienced workers can use the acetone-alcohol mix. Skill is needed to gauge when decolorization is complete.
As the counterstain is also a basic dye, it is possible to replace the CViodine complex in Gram-positive cells with an over-exposure to the counterstain. The counterstain should not be left on the slide for more than 30 s.
| Reporting results|| |
Results and Interpretation (as Described by WHO, 2003)
a. Evaluate the general nature of the smear under low-power magnification (10X).
- Determine if smear has been properly decolorized.
Depending on the source of the specimen, the background should be generally clear or gram negative. If WBCs are present, they should appear completely gram negative. Do not mistake thin crystal/gentian violet precipitate needles for gram-positive bacillus-shaped bacteria.
- Determine if thickness of smear is appropriate. For proper interpretation, areas must be no more than one cell thick with no overlapping of cells.
b. Examine smears prepared from clinical specimens under low power for evidence of inflammation. If appropriate for culture source, note the following:
- Relative amounts of PMNs, mononuclear cells, and RBCs
- Relative amounts of squamous epithelial cells, bacteria consistent with normal microbiota, which may indicate an improperly collected specimen
- Location, shape and arrangements of microorganisms
c. Examine several fields a of the smear under oil immersion for the presence of microorganisms.
- If no microorganisms are seen in a smear of a clinical specimen, report "No microorganisms seen."
- If microorganisms are seen, report relative numbers and describe morphology.
d. Observe predominant shapes of microorganisms overall shape: coccus, coccoid, coccobacillary, bacilli, filamentous, yeast-like
- Appearance of ends; rounded, tapered, flattened, clubbed (swollen), concave, swelling of sides can suggest the presence of spores but can also be caused by vacuoles, marked pleomorphism, or irregular staining.
- Appearance of sides: parallel, ovoid (bulging), concave, irregular
- Nature of axis: straight, curved, spiral
- Pleomorphism (variation in shape): the descriptive term "diptheroid" or "coryneform" is used to describe gram positive bacilli that are pleomorphic, club shaped, or irregularly staining or that have palisading and/or angular arrangements (V and L shapes).
- Branching or cellular extensions
Each laboratory must have SOPs (Standard operating procedures) for reporting results and for all procedures in the Microbiology laboratory. Clinically significant findings should be communicated to the pathologist or called to the attention of the attending physician.
a. Smears of clinical specimens: for urine cultures, examine 20 or more fields. Report as positive if an average of one or more organisms are seen per oil immersion field. This correlates with a colony count of = 10 5 CFU/ml.
- Record relative amounts of observed cells and microorganisms. Commonly used quantitation systems include the following.
- 1+ (<1 per oil immersion field, 100x)
- 2+ (1 per oil immersion field)
- 3+ (2 to 10 per oil immersion field)
- 4+ (predominant or > 10 per oil immersion field)
- Rare (<1 per oil immersion field)
- Few (1 to 5 per oil immersion field)
- Moderate (5 to 10 per oil immersion field)
- Many (>10 per oil immersion field)
- Record the morphology of observed bacteria
| Value of direct smears|| |
Direct Gram stain smears guides the physician on the initial choice of antibiotics, pending the results of culture and sensitivity, Judges Specimen quality, Contributes to the selection of culture media, especially with mixed flora and provides internal quality control when direct smear results are compared to culture results. 
| Further considerations in gram stain|| |
- Use results of Gram stains in conjunction with other clinical and laboratory findings. Use additional procedures (e.g., special stains, direct antigen tests, inclusion of selective media, etc.) to confirm findings suggested by Gram-stained smears.
- Careful adherence to procedure and interpretative criteria is required for accurate results. Accuracy is highly dependent on the training and skill of microscopists.
- Additional staining procedures are recommended for purulent clinical specimens in which no organisms are observed by the Gram stain method.
- Gram stain-positive, culture-negative specimens may be the result of contamination of specimen with normal commensals, or artefacts from staining reagents, Prior use of antimicrobial agents, or failure of organisms to grow under usual culture conditions (media, atmosphere, etc.)
| Summary|| |
The differentiation of bacteria into either the gram-positive or the gram-negative group is fundamental to most bacterial identification systems. This task is usually accomplished through the use of Gram's staining method. Incidentally, the Gram stain is a deceptively simple procedure. Staining can be performed quickly and easily but preparation and interpretation of the smear requires considerable experience and training and can therefore be prone to errors. The use of SOPs and Quality controls are important in the performance of the test.
Reporting Gram stain results of direct smears are more reliable and useful when samples are from sterile sites and may guide the selection of the appropriate initial antibiotic before the results of culture identification and susceptibility testing become available especially for infections like sepsis and meningitis where a delay in the institution of appropriate initial antibiotic results in high mortality. Identification of bacteria such as Acinetobacter spp, Nocardia spp and Actinomycetes spp which can be Gram variable remains a challenging problem The quality of the results and confidence in the reports will improve with the much needed interaction between the Laboratory scientists who generates the results and the Pathologist who interpretes the results in the light of the patients clinical condition with the selection of the most appropriate antibiotic for the management of the patient.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]