Introduction

The Gram’s Stain kit contains crystal violet, iodine, Gram’s stain decolorizer, and safranin. These reagents are part of the Gram staining technique, a differential staining technique used to distinguish Gram-positive and Gram-negative bacteria. The microorganisms that retain the crystal violet dye after the procedure are known as Gram-positive bacteria. They appear a dark purple to a blue color when observed under the microscope. In contrast, the microorganisms that lose the crystal violet dye when the decolorizer is added and take up the secondary stain safranin are known as Gram-negative bacteria. They appear pink to red. Different parts of the cell structure such as flagella, capsules, spores, and granules can be stained using Gram’s stain. Gram stain can be used to diagnose many diseases such as bacterial meningitis, pneumonia, sexually transmitted infections such as gonorrhea, and vaginal infections. It is a rapid, inexpensive, and fairly reliable method for diagnosing these diseases. 

Principle

Gram-positive and Gram-negative bacteria are the two major groups of bacteria. The Gram staining technique is a differential staining technique based on the differential structure of these two bacterial groups’ cellular membranes and cell walls. The cell wall of Gram-positive organisms contains a thick mesh-like peptidoglycan layer that retains the primary crystal violet stain. After iodine is applied, the crystal violet-iodine complex is formed. When the decolorizer is applied, the crystal-violet iodine complex remains, giving the Gram-positive bacteria dark purple to blue color. In contrast, the cell wall of Gram-negative bacteria contains a thin layer of peptidoglycan. Therefore, after the application of crystal violet and iodine, the crystal-violet iodine complex is not trapped within the thin peptidoglycan layer. When the decolorizer is applied, it dehydrates the outer cellular membrane and removes the crystal violet-iodine complex from it, leaving the cells colorless. Safranin, the secondary stain, is then applied to make the Gram-negative cells visible by giving them a red to pink color. 

Apparatus and Equipment 

Bunsen burner, microscope slides, slide rack, microscope.

Procedure 

The following is Gram’s staining protocol for the examination of Gram-positive and Gram-negative bacteria.

Reagents 

1. Crystal violet (primary stain)
2. Iodine (mordant)
3. Gram’s stain decolorizer 
4. Safranin (secondary stain or counterstain)
5. Water

Protocol

1. Prepare a sample smear on a clean microscopic slide and heat fix it.
2. Flood the smear with crystal violet and let it stand for 60 seconds. 
3. Pour the stain off the slide and rinse it with water.
4. Flood the smear with iodine and let it stand for 60 seconds. 
5. Pour the stain off the slide and rinse it with water.
6. Decolorize the slide with Gram’s stain decolorizer for 10-30 seconds until it flows colorless from the slide. (Avoid keeping the decolorizer for too long since it can cause the Gram-positive cells to lose the crystal violet dye as well).
7. Rinse with water.
8. Counterstain the smear with safranin for 20-30 seconds. 
9. Rinse with water and blot dry with absorbent paper.
10. Observe the slide under a microscope under the oil immersion objective. 

Expected Results 

The following are the expected results if the Gram staining procedure is done correctly:

1. Gram-positive bacteria – blue-purple
2. Gram-negative bacteria – pink-red

Applications

Diagnosis of bacterial meningitis with cerebrospinal fluid gram stain (Brizzi, Hines, McGowan, & Shah, 2012).

Laboratory tests are essential for diagnosing bacterial meningitis. Even though bacterial culture is the diagnostic standard, rapid results are not received. Therefore, cerebrospinal fluid (CSF) Gram stain is a good alternative for diagnosis since it is a reliable method and provides rapid results. In this study, the diagnostic accuracy of CSF gram stain was evaluated in neonates and children. Subjects (newborns up to 18 years of age) with lumbar punctures were included. Cytospin slide centrifuge was used to prepare CSF Gram stains. The slides were then stained using conventional Gram staining and examined by light microscopy. Bacteriology cultures were also performed, and isolates were identified using standard procedures. It was observed that 21 specimens met the criteria in which 17 were classified as definite, and 4 were classified as probable bacterial meningitis. CSF Gram stain was positive in 16 of 17 subjects with definite bacterial meningitis with a positive predictive value of 47.1%. The sensitivity was 95.2% for those with definite or probable meningitis. 

Biofilm detection in chronic rhinosinusitis (To’th, Csomor, Sziklai, & Karosi, 2011). 

Chronic rhinosinusitis (CSR) is a common inflammatory disease caused by a variety of pathogens. Among these pathogens, bacterial and fungal biofilms have been implicated in this disease. Diagnosing bacterial and fungal biofilms are difficult because they cannot be cultured or isolated by conventional microbiological protocols and require expensive and time-consuming protocols. Therefore, a rapid, cheap, and reliable method for diagnosing bacterial and fungal biofilms can help. This study used combined hematoxylin-eosin and Gram staining for the histological diagnosis of biofilms. Fifty patients with chronic rhinosinusitis with nasal polyposis (CRS/NP) undergoing endoscopic sinus surgery (ESS) were analyzed. Twelve patients undergoing septoplasty for nasal obstruction without CRS/NP served as the negative control group. Results indicated that in 44 of the 50 patients with CRS/NP, biofilm was detected. In contrast, biofilm was not detected in any of the 12 negative controls. Therefore, combined hematoxylin-eosin and Gram staining is a reliable method for detecting bacterial and fungal biofilms in CRS/NP.

Investigation of microbial profile and antibiotic susceptibility of culture-positive bacterial endophthalmitis (Melo, Bispo, Yu, Pignatari, & Höfling-Lima, 2011).

Infectious endophthalmitis following cataract surgery is a serious condition. Many different types of microorganisms are responsible for causing this condition. Gram-positive bacteria such as coagulase-negative Staphylococcus (CoNS), Staphylococcus aureus, and Streptococcus viridans cause acute postoperative endophthalmitis. Low virulence microorganisms, such as Propionibacterium acnes, some species of Streptococci, and fungi, are usually responsible for late postoperative endophthalmitis. This study assessed the microbial profile and antibiotic susceptibility of culture-positive bacterial endophthalmitis.  Out of 231 patients assessed, 107 showed positive results by Gram stain or culture. One hundred microorganisms were isolated from 97 culture-positive cases, in which 91% were Gram-positive, and 9% were Gram-negative. Therefore, Gram-positive bacteria were the major cause of infectious endophthalmitis. 

Diagnosis of vulvovaginitis (Buyukbayrak et al., 2010).

Vulvovaginal infections need to be properly diagnosed before treatment begins. Although microbiology tests are considered the gold standard for diagnosing vulvovaginitis, it is still difficult to conduct a proper diagnosis since vagina flora contains dozens of species. In this study, current diagnostic clinical and laboratory approaches were compared in 460 women with vulvovaginal discharge complaints. The clinical diagnosis was based on a combination of symptoms and office-based tests such as observation of discharge characteristics on speculum examination, wet mount examination, whiff test, vaginal pH test, and chlamydia rapid antigen test. For microbiological diagnosis, Gram stain and cultures were performed. Out of the 460 patients, only 166 had a microbiological diagnosis while 413 received a clinical diagnosis. The sensitivity, specificity, and positive and negative predictive values of clinical diagnosis over microbiological culture results were 95, 13, 38, 82%. The cultures indicated that Candida species (17.4%) and Gardnerella vaginalis (10.2%) were the most encountered species. Clinical diagnosis results indicated mixed infection (34.1%), bacterial vaginosis (32.4%), and fungal infection (14.1%). Moreover, symptoms did not predict laboratory results. Overall results indicated that laboratory testing is essential in combination with clinical diagnosis to provide an accurate diagnosis. 

Strengths and Limitations 

Strengths 

The Gram stain can be used to distinguish Gram-positive and Gram-negative bacteria. It is a fast, inexpensive, non-invasive, and reliable technique for diagnosing many diseases. Gram stain can help determine the appropriate treatment for infections. Gram stain can test samples of various bodily fluids such as mucus, sputum, and blood. 

Limitations 

It may be difficult to interpret the slides if the microscopic smear preparation is thick and clumped. The decolorizing step should be properly monitored to prevent under-decolorization or over-decolorization. Over-decolorization can result in the Gram-positive cells losing the crystal violet dye, making it impossible to distinguish between Gram-positive and Gram-negative cells. Gram stain should be performed in young, actively growing cultures since older cultures may not have an intact cell wall. Gram stain cannot be used in organisms that don’t have a cell wall like Mycoplasma species and smaller bacteria like Chlamydia and Rickettsia species.

Summary 

1. Gram’s staining technique is a differential staining technique used to distinguish Gram-positive and Gram-negative bacteria.
2. Gram-positive bacteria retain the crystal violet dye after the procedure and appear dark blue to purple. Gram-negative bacteria lose the crystal violet dye and are counterstained with safranin and appear pink to red. 
3. Gram’s staining technique is a rapid, inexpensive, and reliable technique for diagnosing various illnesses and diseases. 

References

Bisen, P.S. (2014). Microbial Staining. Microbes in Practice (pp. 139-155). 

Brizzi, K., Hines, E. M., McGowan, K. L., & Shah, S. S. (2012). Diagnostic accuracy of cerebrospinal fluid gram stain in children with suspected bacterial meningitis. The Pediatric infectious disease journal, 31(2), 195–197. https://doi.org/10.1097/INF.0b013e31823d7b6f

Esim Buyukbayrak, E., Kars, B., Karsidag, A. Y., Karadeniz, B. I., Kaymaz, O., Gencer, S., Pirimoglu, Z. M., Unal, O., & Turan, M. C. (2010). Diagnosis of vulvovaginitis: comparison of clinical and microbiological diagnosis. Archives of gynecology and obstetrics, 282(5), 515–519. https://doi.org/10.1007/s00404-010-1498-x

Melo, G. B., Bispo, P. J., Yu, M. C., Pignatari, A. C., & Höfling-Lima, A. L. (2011). Microbial profile and antibiotic susceptibility of culture-positive bacterial endophthalmitis. Eye (London, England), 25(3), 382–388. https://doi.org/10.1038/eye.2010.236

Tóth, L., Csomor, P., Sziklai, I., & Karosi, T. (2011). Biofilm detection in chronic rhinosinusitis by combined application of hematoxylin-eosin and gram staining. European archives of oto-rhino-laryngology : official journal of the European Federation of Oto-Rhino-Laryngological Societies (EUFOS) : affiliated with the German Society for Oto-Rhino-Laryngology – Head and Neck Surgery, 268(10), 1455–1462. https://doi.org/10.1007/s00405-011-1623-x

Tripathi N, Sapra A. Gram Staining. [Updated 2020 Aug 28]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2020 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK562156/