Interpretation of Gram Stains and Other Common Microbiologic Slide Preparations

  • Authors: Fred Tenover 1, J. V. Hirschmann 2
    Affiliations: 1: Centers for Disease Control, Atlanta, GA; 2: University of Washington, Seattle, Washington
  • Citation: Fred Tenover, J. V. Hirschmann. 2007. Interpretation of gram stains and other common microbiologic slide preparations.
  • Publication Date : February 2007
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With contributions by:

Ellen Jo Baron

Larry Carlson

Carla Clausen

Sharon Hilliar

Greg Raugi

Michael Rinaldi

Jane Schwebke  

Edited and revised by Rebecca Buxton (corresponding author) 
Department of Pathology
University of Utah

Salt Lake City, UT  84132  USA

Originally published in 1990 as Interpretation of Gram Stains and Other Common Microbiologic Slide Preparations by the UpJohn Company, Kalamazoo, Mich. Permission granted to the ASM MicrobeLibrary by Pfizer Inc.


This Atlas was written to help clinicians, microbiologists, and laboratory personnel identify organisms in infected materials stained by techniques commonly used in most clinical laboratories. Because of its time-honored value as a teaching tool for clinical laboratorians and physicians, as much of the Atlas' original print format as possible has been preserved for this electronic version, with some revision in organism nomenclature.

Since microbes grown on culture media may look different from those in the original clinical specimens, only stains of clinical specimens are included. Below find a brief description of the stains used and of microscopic techniques, followed by links to sections of images and descriptions of the pathogens recovered from the following body sources: sputum; urine; vaginal, cervical and urethral secretions; skin; intraabdominal abscesses; spinal fluid; and miscellaneous tissues.

Suspicion that a patient has an infection arises from the clinician's interpretation of the history, the physical examination, certain laboratory test results, and, sometimes, radiographic procedures. The definitive diagnosis typically depends on isolating pathogens on appropriate culture media. However, many patients require prompt treatment before the results of the culture are available. Frequently, presumptive identification of the pathogens can be made from microscopic examination of infected material (e.g., sputum, pus, urine, cerebrospinal fluid), and therapy can be chosen confidently and rationally. Since bacteria cause so many serious treatable infections, this collection focuses on the Gram stain, the most important staining technique for identifying bacteria using light microscopy. However, several other microbiologic slide preparations are also discussed and illustrated.

Unless otherwise noted, each micrograph was taken through an oil-immersion (100x) lens (1,000x total magnification). 

Identification of bacteria by Gram and acid-fast stains 
Because bacterial protoplasm is acidic, a characteristic caused largely by nucleic acids, intact bacteria stain readily with alkaline dyes such as crystal violet, methylene blue, and basic fuchsin, but stain poorly with acidic dyes such as eosin. The most useful microscopic techniques for identifying bacteria are differential stains that employ two or more alkaline dyes to distinguish types of bacteria according to the differences in the chemical composition of their cell walls. The two most important are the Gram stain and the acid-fast stain. 
The Gram stain was originally devised in 1884 by Hans Christian Gram (1853–1938), a Danish physician, to detect bacteria in diseased tissue. For a procedure see the MicrobeLibrary Gram Stain Protocol section. Most bacteria stain either blue (gram positive) or red (gram negative). In both types, crystal violet and iodine form a complex within cells. However, the cell walls of gram-positive organisms have a thick layer of peptidoglycan nearly impermeable to the decolorizing agent, and the blue complex of dyes remains imprisoned within the cell walls. The cell walls of gram-negative bacteria have a thin layer of peptidoglycan and a high lipid concentration that allows alcohol to penetrate and leach out the crystal violet-iodine complex. Those organisms then take up the red counterstain. 
Certain bacteria, especially mycobacteria, have such high lipid concentrations in their cell walls that the organisms stain only feebly with the Gram technique. They are readily visible, however, with acid-fast stains, such as Ziehl-Neelsen or auramine O. With the Ziehl-Neelsen technique those organisms take up the red primary stain, carbol fuschin; unlike other bacteria, their cell walls are relatively impermeable to acid-alcohol and also to the counterstain, methylene blue. Acid-fast organisms, therefore, stain red; the background and other bacteria stain blue. Acid-fast organisms also take up auramine O, a fluorescent microscopy stain that appears yellow on a dark green to black background. 
Some bacteria do not readily incorporate the dyes within their cells and are not easily detected by either Gram or acid-fast stains. Some fungi are visible when stained with Gram stain, but other microorganisms, such as mycoplasmas, rickettsiae, chlamydiae, and viruses, do not take up the dyes or are too small to be seen with light microscopy.
To be visible on a slide, organisms that stain by the Gram method must be present in concentrations of about 104 to 105organisms per milliliter of uncentrifuged fluid. At lower concentrations, the Gram stain of a clinical specimen seldom reveals organisms even if the culture is positive. For example, Gram stains of ascetic fluid from patients with spontaneous bacterial peritonitis are commonly negative despite positive cultures, because the number of bacteria is usually less than 10 per milliliter. 
Examination of Gram stains 
The examiner should first determine whether the stain is adequate (see Sputum–Unacceptable Specimens and Staining Artifacts).In an appropriately stained specimen, the nuclei of neutrophils are red. If the nuclei are blue, the decolorization is insufficient. In thick specimens like sputum, if certain areas are difficult to decolorize, the examiner should look for other areas on the slide where the nuclei are red. If the entire specimen is unsatisfactory, it should be decolorized again with alcohol or acetone-alcohol and then restained with safranin. Repeating the crystal violet and iodine staining is unnecessary. If the decolorization is still inadequate, the slide should be flooded with acid-alcohol (used in acid-fast stains) to remove all stain, and the complete Gram stain procedure should be repeated. If the entire specimen is too thick for adequate decolorization, a new slide with a thinner smear must be prepared. 
If both red and blue organisms are visible, decolorization is satisfactory. Excessive decolorization will cause gram-positive organisms to appear to be gram negative. If only gram-negative organisms are visible and their morphology suggests that they are really gram positive, the entire staining procedure should be repeated. You may refer to the "Comments and Tips" section of the Gram Stain Protocol  for further suggestions.
When examining a properly stained slide, the examiner should note the following characteristics: 
  • the presence of a single type or several types of organisms
  • the predominant type of organism if more than one is present
  • the staining characteristics (gram positive or gram negative)
  • the shape of the organisms, rods (bacilli) or round (cocci)
  • the size of the organisms: small, large, thin, plump
  • the configuration: single organisms, pairs, chains, clumps, clusters, branching
  • the relation to inflammatory cells because some organisms are characteristically inside inflammatory cells (intracellular) or adherent to them 
With that information, the source of the specimen, and a knowledge of the organisms likely to cause infections at the involved site, the examiner can presumptively identify many of the pathogens. However, the genus or species of organisms cannot be predicted reliably from their appearance on a Gram stain. In the case of a patient with acute pneumonia, for example, a properly collected sputum specimen that demonstrates mostly lancet-shaped, gram-positive diplococci will probably grow Streptococcus pneumoniae (pneumoccocci). Although other streptococci can look identical on a Gram stain, pneumococcal pneumonia can be diagnosed confidently because other morphologically similar streptococci only rarely cause acute pulmonary infections. Similarly, to differentiate enteric gram-negative bacilli by their microscopic appearance is difficult. Plump, gram-negative rods in a sputum specimen from a patient with pneumonia strongly suggest that an enteric gram-negative bacillus is the cause. However it cannot be safely assumed to be Klebsiella pneumoniae instead of other enteric bacilli such as Escherichia coli, Enterobacter species, or Serratia marcescens,because these bacteria can also cause lower respiratory tract infections. Clinicians using the Gram stain to help make therapeutic decisions must recognize the limitations of the technique and not overinterpret the findings. 
Links to photographs and legends: 

Examination of Gram Stains of Sputum

Examination of Gram Stains of Urine (6 images)

Examination of Gram Stains of Vaginal Secretions (6 images)

Examination of Gram Stains of Cervical and Urethral Discharges (3 images)

Examination of Gram Stains of Bacterial Skin Infections 

Examination of Gram Stains of Intraabdominal Infections (5 images)

Examination of Gram Stains of Spinal Fluid–Bacterial Meningitis 

Examination of Gram Stains of Miscellaneous Tissue Infections (6 images)

Primary reference:

1.  Tenover, F. C., and J. V. Hirschmann. 1990. Interpretation of Gram stains and other common microbiologic slide preparations. The UpJohn Company, Kalamazoo, Mich.
References for editing and revisions in nomenclature: 

2.  Kwon-Chung, K. J., and J. E. Bennett. 1992. Medical mycology. Lea & Febiger, Philadelphia, Pa.
3.  Murray, P. R., E. J. Baron, J. H. Jorgensen, M. A. Pfaller, and R. H. Yolken. 2003. Manual of clinical microbiology. ASM Press, Washington, D.C.

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