Approach to Gram stain and culture results in the microbiology laboratory

INTRODUCTION — Clinical decisions regarding the management of infections are frequently based on the results of Gram stain and culture. Therefore, it is important that such studies are performed and interpreted correctly.

The quality of the clinical specimen can impact the value of the Gram stain performed. The choice of the specimen sent for Gram stain and culture depends on the site of the infection and the likely pathogens. Good specimen collection should avoid contamination with surrounding colonizing bacteria, provide an adequate volume of material for Gram stain and culture, be collected prior to initiation of antimicrobial therapy and labelled carefully with relevant clinical information, and be transported to the laboratory in a timely manner [1].

Issues relating to the interpretation of Gram stain and culture results are discussed here. Issues relating to the management of specific infections are discussed in detail separately. (See related topics.)

GRAM STAIN — The Gram stain is used to differentiate between different types of bacteria based on the biochemical properties of their cell walls. The method is named after Danish scientist Hans Christian Gram (1853 to 1938), who developed the technique in order to distinguish between two different bacterial causes of pneumonia (Streptococcus pneumoniae and Klebsiella pneumoniae).

Gram staining of clinical specimens (including sterile and nonsterile body fluid specimens, biopsy specimens, and positive culture specimens) is useful for guiding empiric clinical management for bacterial infections pending definitive culture data and/or molecular data. Gram staining also allows visualization of yeast.

Procedure — Performing a Gram stain consists of the following steps [2]:

●Prepare a heat-fixed smear of processed clinical specimen or a bacterial culture on a glass slide.

●Apply the primary stain (crystal violet) to the slide; rinse with water.

●Immerse the slide in mordant (Gram's iodine).

●Perform rapid decolorization (a matter of seconds) with acetone or alcohol. The decolorization step is critical, and it must be timed correctly; if the decolorizing agent is left on too long, crystal violet stain will be removed from gram-positive cells as well as gram-negative cells.

●Counterstain with safranin, basic fuchsin, or neutral red. Basic fuchsin (present in dilute carbol fuchsin) stains many gram-negative bacteria more intensely than the other two stains.

Specimens

Sterile sites — Sterile sites refer to anatomic sites in which bacterial organisms are not present in the absence of infection. Fluid and/or tissue from these sites should be collected under sterile conditions (examples include cerebrospinal fluid, pleural fluid, pericardial fluid, synovial fluid, and peritoneal fluid). Colonizing bacteria rarely contaminate specimens from sterile sites in sufficient quantities to be detected on Gram stain [3].

Any bacteria detected on Gram stain of a sterile site specimen should be considered significant, although a negative Gram stain does not exclude infection [3]. For sterile body fluids, the sensitivity of Gram stain may be increased by cytospin centrifugation prior to staining (particularly cerebrospinal fluid) [4,5].

Nonsterile sites — Nonsterile sites refer to anatomic sites in which colonizing organisms ("normal flora") may be present. Specimens from these sites are typically contaminated with colonizing bacteria ("normal flora"). Examples include sputum, throat swabs, wound swabs, and genital swabs (picture 1).

These specimens also generally contain human cells such as epithelial cells and white blood cells. Human cells are stained by the safranin (or carbol fuchsin) counterstain [6]. Human cells present in these specimens (such as neutrophils, lymphocytes, and epithelial cells) are reported "semi-quantitatively" (eg, 1+ to 4+ or rare-few-moderate-abundant). This information can be used to assess the quality of the specimen and whether inflammation is present. Note that white cells may be absent in the presence of infection if the patient is neutropenic. In addition, the presence of gram-positive bacilli in the absence of white cells should raise suspicion for gas gangrene.

A high-quality sputum specimen has high numbers of white cells and few epithelial cells; a low-quality specimen has high numbers of epithelial cells and low numbers of white cells. Low-quality specimens may be rejected by the laboratory as unfit for culture; criteria for rejecting specimens differ between laboratories [7,8].

Interpretation of Gram stains from nonsterile sites requires experience and knowledge of the normal expected flora. A sputum specimen with a large number of bacteria of different morphologies may be consistent with normal oral colonizing bacteria. On the other hand, a high-quality sputum specimen with large numbers of bacteria present on Gram stain demonstrating the typical morphology of a possible respiratory tract pathogen may be a useful clue as to the etiology of infection [9].

INTERPRETING GRAM STAIN RESULTS — In general, Gram staining allows categorization of observed microorganisms into two major groups: gram positive and gram negative. Gram-positive bacteria retain crystal violet and appear purple with Gram staining; gram-negative bacteria do not retain crystal violet but take up safranin counterstain and thus appear pink. A few gram-negative organisms tend to stain faintly with safranin (such as Campylobacter species), and thus an alternative counterstain such as carbol fuchsin may be preferred [10].

Some organisms are gram variable (ie, they may stain either negative or positive). Common gram-variable organisms observed in clinical specimens include Gardnerella vaginalis and Mobiluncus species. These organisms are associated with bacterial vaginosis and are technically classified as "gram positive." In addition, some gram-negative organisms such as Acinetobacter species tend to retain crystal violet and may sometimes appear gram variable.

Some bacterial species cannot be visualized by Gram stain because they either lack a cell wall (eg, Mycoplasma species) or because their cell wall structure does not retain Gram stain reagents (eg, Chlamydia and Mycobacterium species; the latter may have a "beaded" gram-positive appearance) [10,11]. Some nonbacterial species also stain purple with Gram stain (such as Candida species) (picture 2).

Gram-stained bacteria are described according to the morphology of individual bacteria (eg, spherical "cocci," rod-shaped "bacilli," "coccobacilli," and curved bacteria), their arrangement (eg, "chains" or "clusters"), and their size. The morphology of individual bacteria may occasionally be atypical if the patient has received antibiotics prior to specimen collection. For example, some gram-negative bacteria may become longer and more filamentous [12].

For clinical specimens collected from sterile sites, semi-quantification of the number of organisms present in the Gram stain may be useful. Results may be reported as occasional, small, moderate, or large numbers of bacteria present or using a grading system of 1+ to 3+. The presence of any bacteria in these specimens should be considered significant. The absence of bacteria is also important to report. This approach is less useful when clinical specimens are collected from nonsterile sites, as commensal bacteria may not be easily distinguishable from pathogenic bacteria.

Gram-positive organisms

Gram-positive cocci — An approach to gram-positive cocci is summarized in the algorithm (algorithm 1).

●Clusters - Gram-positive cocci in clusters usually indicate Staphylococcus species, although Gram staining cannot differentiate Staphylococcus aureus from other staphylococcal species. Other species that manifest as gram-positive cocci in clusters and occasionally appear in clinical specimens include Micrococcus, Dermacoccus, Alloiococcus, Rothia, and Aerococcus species (picture 3 and picture 4 and picture 5) [13]. The coagulase test may be used for differentiating S. aureus from other staphylococcal species. Yeast forms also stain gram positive and should not be mistaken for gram-positive bacteria (picture 2). Yeast cells are larger in size than bacteria, are single cells, and may having budding or pseudohyphae present.

●Chains - Gram-positive cocci in chains usually indicate Streptococcus or Enterococcus species (picture 6). Gram staining cannot distinguish between viridans streptococci, hemolytic streptococci, or enterococci; in some cases, the pattern of hemolysis may be used to distinguish these organisms (algorithm 1). (See 'Types of hemolysis' below.)

Other species manifesting as gram-positive cocci in chains that very occasionally appear in clinical specimens include Leuconostoc, Abiotrophia, Granulicatella, Pediococcus, and Gemella species other than G. haemolysans. Anaerobes such as Micromonas, Finegoldia, Peptostreptococcus, and Peptococcus spp also have this appearance [13].

The finding of gram-positive diplococci (spheres in pairs) is pathognomonic for S. pneumoniae; this organism may also be arranged in short chains. Individual bacteria classically appear slightly elongated and arranged end to end (picture 7 and picture 8 and picture 9) [10,14].

Gram-positive bacilli (rods) — An approach to gram-positive bacilli is summarized in the algorithm (algorithm 2).

●Large - Large, square-ended gram-positive rods usually indicate anaerobic Clostridium species or aerobic Bacillus or Lactobacillus species. These bacilli are typically large and have a "box-car" shape with squared-off ends and parallel sides (picture 10 and picture 11) [10,14,15]. Paenibacillus and Brevibacillus may stain gram positive, gram negative, or gram variable. The presence of gram-positive or gram-variable bacilli in soft tissue specimens without visible white cells should raise suspicion for gas gangrene.

●Medium - Medium-length gram-positive rods include Corynebacterium species [10,15]; Cutibacterium (formerly Propionibacterium) and Listeria species may also have this appearance (picture 12) [14]. Others include Lactobacillus, Bifidobacterium, and Eubacterium. Corynebacterium species in fluid media exhibit a typical club-shaped morphology with clumping of cells, the so-called "Chinese letters" appearance (picture 13).

●Small - Small, palisading gram-positive rods include Corynebacterium, Cutibacterium, Listeria, Gardnerella (gram variable), and Rhodococcus. These may be pleomorphic or "club shaped" and arranged in parallel (palisading) formations and/or in arrangements resembling "Chinese letters" (picture 13) [10,15]. This appearance is typical of Corynebacterium species; Cutibacterium and Listeria species may also have this appearance [14]. Small gram-positive coccobacilli classically indicates Listeria species (picture 14 and movie 1), although the Gram stain appearance of this organism may be variable [10,15].

●Branching - Branching gram-positive bacilli usually indicate either the aerobic Nocardia species or the anaerobic Actinomyces species (picture 15 and picture 16). Streptomyces and Cutibacterium may also have this appearance [10,15].

Gram-negative organisms — An approach to gram-negative organisms is summarized in the algorithm (algorithm 3).

Gram-negative cocci — Gram-negative cocci usually indicate Neisseria meningitidis or Neisseria gonorrhoeae. Typically, these species are arranged as pairs ("diplococci") (picture 17). Other gram-negative cocci include Moraxella catarrhalis, other Neisseria species, and the anaerobic Veillonella species (picture 18) [10,15]. Acinetobacter species may also have this appearance, though it may also retain crystal violet and thus appear gram positive [14].

Gram-negative bacilli (rods) — Gram stain cannot reliably distinguish between most aerobic and anaerobic species of gram-negative bacilli [10,15]. However, some reports describe gram-negative bacilli as fusiform, curved, or small gram-negative coccobacilli (picture 19 and picture 20 and picture 21).

●Medium to long, plump - Medium-to-long, plump gram-negative rods include Enterobacteriaceae (Escherichia, Klebsiella, Enterobacter, etc), Bacteroides, Prevotella, Fusobacterium, and other organisms (picture 22).

●Medium to long, thin - Medium-to-long, thin gram-negative rods include Pseudomonas, Enterobacteriaceae (Escherichia, Klebsiella, Enterobacter, etc), Bacteroides, Capnocytophaga, Prevotella, Fusobacterium, and other organisms (picture 23). Gram-negative bacilli with tapered ends (a "fusiform" or filamentous appearance) include Fusobacterium nucleatum, an anaerobe, and Capnocytophaga species (picture 24) [10,15]. Some gram-negative bacteria may become longer and more filamentous in the setting of antibiotic therapy [12].

●Short to long, pleomorphic - Pleomorphic, short-to-long, sometimes vacuolated gram-negative rods include Bacteroides, Fusobacterium, Enterobacteriaceae (Escherichia, Klebsiella, Enterobacter, etc), Pseudomonas, and other organisms (picture 25).

●Tiny - Tiny gram-negative rods (0.2 to 3 micron), often appearing coccobacillary, correspond to a number of organisms including Haemophilus, Acinetobacter, Moraxella, Prevotella, and Porphyromonas [10,15]. Haemophilus species may also appear as long filaments (picture 26 and picture 27) [14].

●Curved - Curved gram-negative rods (0.2 to 0.8 microns wide by 0.5 to 5 microns long) are a typical appearance of Vibrio species. Campylobacter and Helicobacter species may also appear curved or "S shaped" (picture 28) [10,15].

Candida — Candida is a nonbacterial species that stains purple with Gram stain. Long "pseudohyphae" (elongated structures) may be present as well as daughter yeast cells "budding" from larger parent yeast cells (picture 29) [10,15].

CULTURE

Approach — Traditional diagnostic microbiology techniques rely on the growth of organisms on appropriate culture media. In general, specimens from sterile sites may be inoculated onto enriched all-purpose media such as blood or chocolate agar; most ordinary bacterial pathogens grow readily on these nonselective solid media. Specimens from nonsterile sites that contain mixed microbial flora should also be inoculated onto additional plates such as MacConkey or other selective media. Selective media are used to inhibit the growth of commensal colonizing bacteria.

After bacteria have been isolated on solid agar media (for most organisms, this occurs within 24 to 48 hours), colony morphology, growth requirements, and rapid bench tests may facilitate preliminary or presumptive identification. Confirmation of species identification, if in pure culture, usually can be done after 24 to 48 hours incubation of the cultures directly from colonies on the media by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF). Colonies of interest selected from mixed cultures (such as those typically recovered from nonsterile site specimens) must be "subcultured" prior to identification in order to obtain a pure culture (picture 30). (See 'MALDI-TOF' below.)

Bacteria initially cultured in broth medium (eg, blood cultures) are evaluated by Gram stain, followed by inoculation of blood culture broth onto solid media. Direct identification of isolates from broth cultures may be attempted by MALDI-TOF [16-18].

Growth conditions — Important growth conditions include temperature and atmospheric conditions. Most bacterial cultures are incubated at 35 to 37°C. Atmospheric conditions used in the microbiology laboratory include aerobic, anaerobic, and 5 percent CO₂. Many facultative anaerobic organisms such as streptococci grow well under anaerobic conditions. In general, however, anaerobic organisms do not grow readily under aerobic conditions. Capnocytophaga species require 5 percent CO₂ for optimal growth ("capnophilic organisms") [10,15]. Other microorganisms may also require specialized atmospheric conditions. For example, Campylobacter species grow most readily under "microaerophilic conditions" [10,14,15].

Culture media

Blood agar — Blood agar supports the growth of many common human bacterial pathogens (with the exception of Haemophilus influenzae). Blood agar culture plates are also useful for observing hemolysis (the ability of bacterial colonies to break down red blood cells).

Types of hemolysis — The presence of and type of hemolysis is useful for classifying streptococcal species [10] (algorithm 1).

●Alpha hemolysis (incomplete or partial hemolysis) is caused by bacterial production of hydrogen peroxide, which in turn oxidizes hemoglobin (red) to methemoglobin (green) (picture 31). S. pneumoniae and viridans streptococci display alpha hemolysis.

●Beta hemolysis (complete red cell lysis) results in transparency in the normally red agar media. This transparency is typically present around and under the colonies (picture 32 and picture 33). Streptococcus pyogenes and Listeria typically display beta hemolysis. Some weakly beta-hemolytic species (eg, S. agalactiae and C. perfringens) may induce intense beta hemolysis when grown concurrently with S. aureus.

●Gamma hemolysis (no hemolysis) results in no change in the agar color around and under the colony. Enterococcus faecalis (formerly group D Streptococcus) displays gamma hemolysis.

Hemolytic streptococci are further categorized by Lancefield grouping. (See 'Bench tests' below.)

Chocolate agar — Chocolate agar is named for its brown color. It contains lysed red blood cells that have released growth factors hemin (X factor) and NAD (V factor). These growth factors are necessary for the in vitro growth of H. influenzae (picture 34 and picture 35) [10,14,15].

MacConkey agar — MacConkey agar is a selective medium designed to detect gram-negative bacteria. Bile salts in MacConkey agar inhibit growth of gram-positive bacteria. Lactose-fermenting organisms produce pink colonies when grown on this media; nonlactose-fermenting organisms produce colorless colonies (picture 36). Lactose-fermenting organisms include Escherichia coli, Enterobacter, and Klebsiella. Nonlactose-fermenting organisms include Pseudomonas, Proteus, Salmonella, and Shigella.

Certain gram-negative species do not grow on MacConkey agar (so called "fastidious" gram negatives). These include Bordetella, Brucella, Campylobacter, Haemophilus, Legionella, Pasteurella, Acinetobacter, and Eikenella.

Selective media — Selective media are primarily used for cultures of specimens collected from nonsterile sites. These selective media can help detect specific pathogenic organisms growing concurrently with normal colonizing bacteria. These selective media typically contain antibiotics. Pathogens that may be identified with selective media include Bordetella pertussis, Salmonella species, Shigella species, N. gonorrhoeae, and Legionella pneumophila (picture 37).

Colony morphology — Colony morphology for a given organism may vary according to the agar medium used and the growth conditions. Colonies are described based on their texture, size, shape, and color [10,14,15]:

●K. pneumoniae colonies frequently appear "mucoid" on blood agar.

●M. catarrhalis produces "dry" colonies that can be pushed across the agar.

●Yersinia enterocolitica manifests as "pinpoint" colonies on MacConkey agar.

●Proteus species produce large "swarming" colonies on MacConkey agar.

●S. pneumoniae produces colonies with a central depression such that they resemble pieces used for the game of drafts ("draftsmen" appearance).

●Some strains of Serratia marcescens produce non-diffusable red pigment, prodigiosin, or 2-methyl-3-amyl-6-methoxyprodigiosene.

Several common pathogens have distinctive odors (although "sniffing" cultures is generally not encouraged):

●Anaerobic organisms are typically foul smelling.

●Pseudomonas aeruginosa has a "grape-like" or "fruity" odor.

●H. influenzae is said to have a "mousy" odor.

●Streptococcus intermedius group (also known as the Streptococcus milleri group or Streptococcus anginosus group) has a distinctive "caramel" or "butterscotch" odor [10,14,15].

●Eikenella has a "bleach-like" odor.

Bench tests — The following biochemical tests are commonly used to determine the genus and/or the species of a pathogen grown in culture.

●Coagulase test - The coagulase test is used to differentiate S. aureus from other staphylococci (picture 38). The test typically takes ≤4 hours but may take up to 24 hours. In many laboratories, the coagulase test has been replaced by commercially available rapid latex agglutination tests, which take several minutes and may be used for presumptive identification of S. aureus [10].

●Catalase test - The catalase test is used to distinguish staphylococci from streptococci and enterococci. Catalase production is indicated by the presence of oxygen bubbles when bacterial colonies are exposed to hydrogen peroxide (picture 39) [10]. This test can have false-positive results if colonies grown on blood agar are tested.

●Oxidase test - The oxidase test is used to differentiate gram-negative bacilli based on their production of certain cytochrome c oxidases. Bacterial colonies are inoculated onto strips impregnated with oxidase reagent. This reagent appears purple when oxidized and colorless when reduced; a positive result is indicated by a purple color change that is detectable within a few seconds.

Oxidase-positive organisms include P. aeruginosa, Pasteurella multocida, Vibrio, and Aeromonas species. Oxidase-negative organisms include Enterobacteriaceae (such as E. coli, K. pneumoniae, Enterobacter cloacae, Serratia spp) and Acinetobacter species [10].

●Lancefield grouping - Lancefield grouping of hemolytic streptococci groups are based on specific carbohydrates in the bacterial cell wall that allow agglutination with particular antisera. Not all streptococci can be grouped; some species such as S. pneumoniae do not express Lancefield antigens.

Lancefield grouping is usually used to identify beta-hemolytic streptococci but may also be used to help identify other streptococci and enterococci. For example, Enterococcus species are usually group D; S. anginosus group are frequently group F but may be groups A, C, or G [10,15].

●Bile solubility - Bile solubility testing is useful for rapid differentiation of S. pneumoniae (which are bile soluble) from other alpha-hemolytic streptococci [10,15].

●PYR hydrolysis - Seldomly, pyrrolidonyl arylamidase (PYR) hydrolysis is used for further identification of gram-positive cocci in chains. A positive test suggests S. pyogenes, Enterococcus, or Abiotrophia/Granulicatella [10,15].

MALDI-TOF — Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) is a mass spectrometry tool that allows rapid and accurate identification of genus and species for a wide range of gram-negative and gram-positive bacteria as soon as an organism is available in a pure culture on solid media [16-18]. MALDI-TOF detects a characteristic spectrum for each species that can be matched against a large database of spectra within the instrument [19]. For less common organisms, the MALDI-TOF result may not be conclusive, and additional bench tests or molecular tests may be required.

CORRELATING RESULTS — Gram stain findings must be correlated with culture results. A Gram stain demonstrating multiple bacterial species together with only one or two aerobic species isolated in culture, for example, can be an important clue to the presence of anaerobes. If the Gram stain is positive and the cultures are negative, possibilities include the presence of anaerobes, false-negative results reflecting prior antibiotic therapy, or a fastidious organism that cannot grow on routine media.

SUMMARY

●Procedure − The Gram stain is a laboratory technique used to differentiate between different types of bacteria based on the biochemical properties of their cell walls. (See 'Procedure' above.)

●Specimens − The presence of bacteria in Gram stains of specimens collected from sterile sites should almost always be considered a significant finding. However, Gram stains of specimens from nonsterile sites must be interpreted with care. For these specimens, the number and types of human cells present provides information about the quality of the specimen and the presence of inflammation. (See 'Sterile sites' above and 'Nonsterile sites' above.)

●Interpreting Gram stain results (See 'Interpreting Gram stain results' above.)

•Approaches to gram-positive and gram-negative organisms are summarized in the algorithms (algorithm 1 and algorithm 3 and algorithm 2).

•Gram staining allows categorization into two major groups: gram positive and gram negative. Gram-positive bacteria retain crystal violet and appear purple with Gram staining, whereas Gram-negative bacteria do not retain crystal violet but take up safranin counterstain and thus appear pink.

•Gram-stained bacteria are described according to the morphology of individual bacteria (eg, spherical "cocci" or rod-shaped "bacilli"), their arrangement (eg, "chains" or "clusters"), and size.

●Culture (See 'Culture' above.)

•Inoculation − Specimens from sterile sites should be inoculated onto enriched all-purpose media such as blood or chocolate agar; most ordinary bacterial pathogens grow readily on these nonselective solid media. Specimens from nonsterile sites that might contain mixed microbial flora should also be inoculated onto selective media such as MacConkey agar or other selective media. (See 'Approach' above and 'Culture media' above.)

•Colony morphology − A number of common organisms have characteristic morphologies; the colonial morphology of a given organism may vary according to the agar medium used and the growth conditions. Colonies are described based on their texture, size, shape, and color. Several common pathogens have distinctive odors (although sniffing cultures is generally not encouraged). (See 'Colony morphology' above.)

•Bench tests − Bench tests are biochemical tools used for determining the species of a pathogen grown in culture. Common bench tests include the coagulase, catalase, and oxidase tests. (See 'Bench tests' above.)