|What is the Value of a Direct Smear?|
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.
|Determine the Quality of a Sputum Specimen|
Prior to culturing a sputum specimen, a Gram stain should be performed to evaluate the quality of the specimen. One of two criteria are currently used to determine if the specimen is contaminated with oral flora organisms, which would make the specimen unsuitable for culture. One criterion states that the sputum specimen should be rejected if 25 or more squamous epithelial cells/low power field (SQE/LPF) are observed. The second criterion recommends a cutoff of more than 10 SQE/LPF. In either case, a minimum of 20 LPFs should be observed. Review and follow your laboratory's criteria.The low power microscopic field on the right is representative of 20 microscopic fields that were reviewed on this Gram-stained preparation of sputum. This specimen would be considered unacceptable for culture. If the specimen is determined to be a sub-optimal specimen, the clinician should be contacted and a request should be made for a new specimen. It is important to communicate that culturing the specimen that was provided will not yield useful information about the possible pathogens from the lower respiratory tract. If the specimen is determined to be a good quality, lower respiratory tract specimen, continue to examine the slide under oil immersion (1000X) magnification for bacteria, yeast, and polymorphonuclear white blood cells (PMNs) and proceed with culturing the specimen.
|Determine the Quality of a Urine Specimen Submitted for Culture|
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."
|Correlate Direct Smear Results With Culture Results|
Correlation between a direct Gram-stained smear of the specimen and what grows out in culture should always be attempted. However, some bacterial organisms may appear differently when viewed in direct clinical specimens versus isolates growing on solid or in liquid culture media. For example, if gram-negative organisms are surrounded by large amounts of cellular material in the direct smear, the cellular material may totally or partially prevent decolorization of the bacteria so that the gram-negative bacteria growing in culture may appear gram-positive or gram-variable on direct smear.
|Provide the Clinician With Same-Day Information Regarding Possible Pathogens|
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).
|Match the Gram stain reaction for each of the following nonbacterial elements.||View Page|
|Size and Appearance of Nonbacterial Cellular Elements on Gram Stained Smears|
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.
|What nonbacterial structures are present in this microscopic field of a Gram-stained smear? (Choose ALL that apply)||View Page|
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
|What are the structures indicated by the arrows in this field from a Gram-stained smear?||View Page|
|Reporting Microscopic Findings|
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)
|What color is the background material in a Gram-stained smear that has been adequately decolorized?||View Page|
|What is the MINIMUM number of fields that should be examined before reporting a Gram-stained direct smear?||View Page|
|Which of the following statements is true regarding the Gram-stained smear that is represented by this image?||View Page|