Gram-stained Information and Courses from MediaLab, Inc.

Gram stain is used primarily as a differential stain for bacteria, although it will also stain most fungi (especially yeasts) and some parasites, including Strongyloides and Trichomonas. The Gram stain procedure is commonly performed on direct smears of clinical specimens and on smears from cultures. This course will focus on Gram-stained direct smears. A direct smear from a clinical specimen can be used to: Judge the quality of the specimen. Provide the clinician with same-day information regarding possible pathogenic organisms, pending results of culture and sensitivity. Contribute to selection of culture media, especially with mixed flora. Provide internal quality control when direct smear results are compared to culture results.

The presence of many squamous epithelial cells (SQEs) also indicates a poorly collected urine specimen. If many SQEs are noted upon microscopic examination, the specimen should be recollected. The patient must be instructed how to collect a midstream, clean catch specimen. A Gram stain of a fresh, midstream urine sample would provide information that could help the physician decide whether to prescribe an antibiotic and the choice of antibiotic based on gram-reaction of the bacteria. Examine a Gram-stained slide made from a drop of uncentrifuged urine under oil immersion (1000X) magnification. If more than one bacterial organism is observed per oil immersion field, it can be determined that the quantity of bacteria is >105 colony forming units (CFU) per mL, and the patient probably has a urinary tract infection (UTI). The Gram stain reaction would also be important. Most bacteria that cause UTIs are gram-negative Enterobacteriaceae. A Gram stain report in this case would be "gram-negative bacilli consistent with quantity >105 CFU/mL."

Cultures often require 24 or more hours before a pathogen can be recovered. A Gram stain can give preliminary information about the type of bacterial and/or fungal organisms that are present. A rapid diagnosis of bacterial meningitis, made after examining a gram-stained smear of the patient's cerebrospinal fluid, allows the physician to begin treatment immediately. Intracellular gram-negative diplococci observed in a male urethral specimen may be confirmatory of the diagnosis of gonorrhea. Cultures may not even be needed unless susceptibilities are required. (In the female genital specimen, the presence of gram-negative diplococci is not specific enough to confirm the diagnosis, and a culture or other confirmatory testing must be performed).

Type of Cell Average Size Image Comments Epithelial cells 25 µm Appear pink/red on Gram stained smear. Larger than white blood cells. Have a single nucleus. They are an indication of a suboptimal or unacceptable specimen if present in large numbers in sputum specimens, tracheal or endotracheal aspirates, or in urine specimens. White blood cells 12 µm Appear pink/red on Gram stained smear. Most often, polymorphonuclear white blood cells (PMNs). White blood cells indicate inflammation and possible infection. The direct smear examination should focus within and around these cells. Hyphae/pseudohyphae Varies Appear blue on Gram stained smear. Hyphae are tubular filamentous fungal elements, which may show branching or intertwining. Pseudohyphae are multiple buds of yeast that do not detach, thereby forming chains. Yeast 7 µm Appear blue on Gram stained smear. Round to oval, often budding. About the same size as red blood cells. Generally much larger than bacteria. A few yeast may be present as normal flora in upper respiratory tract or genital tract. They may be significant if they predominate, or if budding yeast forms are seen. Red blood cells 7µm Appear red on Gram stained smear Not usually considered a significant finding.

Bacteria may have a very distinctive appearance when viewed on a Gram-stained smear. Distinct characteristics are not as evident on direct smears as they are on Gram stains of colonies and/or stains from broth cultures, but these shapes can be identified on direct smears. Cocci: Round or oval Bacilli: Rod-shapedGram-positive cocci (GPC): Singly and in clusters (May indicate staphylococci) In pairs and chains (May indicate streptococci)GPC can be seen in clusters and chains in image 1 Image 1

Gram-stained direct smears are examined using the oil immersion objective of the microscope (total magnification =1000x). The quantity and type of bacteria and nonbacterial cellular elements present are recorded. A minimum of ten microscopic fields should be examined before reporting the Gram stain result. Organisms and other cells that are observed on a Gram-stained smear should be reported with as much description as possible.In addition to staining reaction, the shape of the organisms should be reported (e.g., cocci or bacilli). It may also be useful to report the cellular arrangement of microorganisms on the smear, although this is more often reported from a smear that is made from culture rather than a direct smear. Cellular arrangements may be described as: Single cells Pairs Tetrads Chains Clusters Budding (in the case of yeast)