148 Postgrad Med J 2001;77:148–156 REVIEWS Postgrad Med J: first published as 10.1136/pmj.77.905.148 on 1 March 2001. Downloaded from Use of the diagnostic bacteriology laboratory: a practical review for the clinician W J Steinbach, A K Shetty Lucile Salter Packard Children’s Hospital at EVective utilisation and understanding of the Stanford, Stanford Box 1: Gram stain technique University School of clinical bacteriology laboratory can greatly aid Medicine, 725 Welch in the diagnosis of infectious diseases. Al- (1) Air dry specimen and fix with Road, Palo Alto, though described more than a century ago, the methanol or heat. California, USA 94304, Gram stain remains the most frequently used (2) Add crystal violet stain. USA rapid diagnostic test, and in conjunction with W J Steinbach various biochemical tests is the cornerstone of (3) Rinse with water to wash unbound A K Shetty the clinical laboratory. First described by Dan- dye, add mordant (for example, iodine: 12 potassium iodide). Correspondence to: ish pathologist Christian Gram in 1884 and Dr Steinbach later slightly modified, the Gram stain easily (4) After waiting 30–60 seconds, rinse with [email protected] divides bacteria into two groups, Gram positive water. Submitted 27 March 2000 and Gram negative, on the basis of their cell (5) Add decolorising solvent (ethanol or Accepted 5 June 2000 wall and cell membrane permeability to acetone) to remove unbound dye. Growth on artificial medium Obligate intracellular (6) Counterstain with safranin. Chlamydia Legionella Gram positive bacteria stain blue Coxiella Ehrlichia Rickettsia (retained crystal violet). Gram negative bacteria stain red (decolorised and then counterstained). No cell wall Spirochetes Mycoplasma Borrelia Ureaplasma Leptospira Spirillum organic solvents (box 1).3–5 Information derived Treponema from a Gram stain and several simple bio- Aerobic chemical tests can be enormous, often provid- Cocci Bacilli Coccobacilli ing a presumptive diagnosis and significantly Gram positive Gram negative Gram positive Gram negative Gram negative Enterobacteriaceae: influencing patient care. Unfortunately the Enterococcus Moraxella Arcanobacterium Citrobacter Bartonella training for correctly interpreting the Gram http://pmj.bmj.com/ 6 Staphylococcus Neisseria Bacillus Edwardsiella Bordetella stain is disappearing. The goal of this article is Streptococcus Corynebacterium Enterobacter Brucella to review the utility of the clinical bacteriology Erysipelothrix Escherichia Campylobacter Gardnerella Hafnia Eikenella laboratory and discuss its role in the diagnosis Lactobacillus Klebsiella Franciscella of common clinical pathogens. Listeria Morganella Haemophilus Mycobacterium Proteus Helicobacter Gram stain basics Nocardia Providencia Kingella Salmonella The Gram stain classifies bacteria (fig 1) Serratia phenotypically based on diVerences in cell wall on October 1, 2021 by guest. Protected copyright. Shigella thickness with diVering glycosaminopeptide Yersinia and lipoprotein compositions: Gram positive bacteria have a peptidoglycan layer 10–15 Non-enterobacteriaceae: (Fermentative) times thicker than Gram negative bacteria. The Aeromonas cell wall, synonymous with the peptidoglycan Pasteurella layer, is a rigid framework of cross linked pep- Plesiomonas tidoglycan forming the outermost component Vibrio of the cell. The more complex Gram negative (Non-fermentative) bacteria also have an outer membrane beyond Acinetobacter the peptidoglycan layer that consists of li- Alcaligenes popolysaccharide (endotoxin), lipoprotein, and Burkholderia Flavobacterium phospholipids. In some Gram negative species Pseudomonas there also exists a periplasmic space between Stenotrophomonas Anaerobic the outer membrane and the inner cytoplasmic Cocci Bacilli membrane with â-lactamases that degrade Gram positive Gram negative Gram positive Gram negative â-lactam antibiotics. The present hypothesis for the mechanism of Peptococcus Veillonella Actinomyces Bacteroides the Gram stain states the cell wall acts as a Peptostreptococcus Bifidobacterium Fusobacterium Clostridium Porphyromonas physical permeability barrier restricting diVu- 7 Eubacterium Prevotella sion of the stain complex, and any micro- Propionibacterium organism with a cell wall able to retard the Figure 1 Classification of clinically significant bacteria by genus. eZux of the crystal violet-iodine complex www.postgradmedj.com Use of the diagnostic bacteriology laboratory 149 should be Gram positive. The mechanism fur- cells to stain Gram negatively. The age of the Postgrad Med J: first published as 10.1136/pmj.77.905.148 on 1 March 2001. Downloaded from ther implies that solvent decolorisation causes culture also influences the degree of Gram significant damage to the cell surfaces of Gram positivity, with cells 48 hours old sometimes negative bacteria, and only limited damage to more Gram positive than younger cells.8 Gram positive bacteria. This suggests Gram Several bacteria are unable to be Gram negative bacteria are more “leaky”, causing stained for a variety of reasons. Mycobacteria these thin walled lipid-rich cells to lose their and nocardia have a high concentration of lip- crystal violet stain and appear red from the ids called mycolic acids in their cell walls and counterstain. Gram positive cells, thick walled are “acid-fast” because they resist decolorisa- and lipid-poor, appear blue from retaining the tion with an organic solvent. The spirochetes original crystal violet. (treponema, borrelia, leptospira, spirillum) are too thin and are best seen with darkfield Gram stain utility microscopy. Legionella, rickettsia, coxiella, Gram stain interpretation gives immediate ehrlichia, and chlamydiae are primarily intra- information regarding the presence or absence cellular and although possess outer and inner of bacterial disease and can guide initial membranes similar to Gram negative bacteria, antibiotic treatment. Additionally, epithelial lack a peptidoglycan layer to take up a Gram and inflammatory cells are stained in a Gram stain adequately. Mycoplasma and ureaplasma stain, thus providing information about the do not have a cell wall to absorb the stain. host immune response and quality of the specimen. A well prepared sample can show- Specific bacteria case the organism’s colour, size, shape, and GRAM POSITIVE COCCI arrangement, allowing cellular morphology to The two principal medically important genera further separate bacteria into four major are staphylococcus and streptococcus, ar- groups. Cocci are spherical or oval, bacilli are ranged in irregular grapelike clusters and rod-like or cylindrical, vibrios are comma-like chains, respectively (fig 2). The orientation and degree of attachment at the time of cell division or curved, and spirochetes are flexible (spirilla determines the type of arrangement: staphylo- if rigid) and helical. Additionally, coccobacilli cocci divide in three planes while streptococci are unusually short bacilli, and fusiform bacilli divide in only one plane. Enterococci are are bacilli with tapered ends. closely related to the streptococci yet are now known to be phylogenetically distinct and Limitations therefore comprise their own genus. The Several substances have been shown to convert enzyme catalase, which degrades hydrogen Gram staining results. Ultraviolet light, antibi- peroxide to oxygen and water, diVerentiates otics, prolonged heat fixation, crushing of catalase positive staphylococci from catalase unprotected cells on a slide, or autolysis by negative streptococci and enterococci. enzymes such as ribonucleases or lysozymes have all been shown to cause Gram positive STAPHYLOCOCCI Catalase Staphylococci are a major component of the http://pmj.bmj.com/ normal human flora and the presence of Positive Negative coagulase, which accelerates the formation of a Staphylococcus fibrin clot from fibrinogen, diVerentiates the species. Although there are 29 species of Coagulase coagulase negative staphylococci, most clinical Positive Negative isolates are either Staphylococcus epidermidis or S aureus Novobicin Staphylococcus saprophyticus. Staphylococcus epi- dermidis is part of the normal skin flora. on October 1, 2021 by guest. Protected copyright. Sensitive Resistant Although often occurring as a contaminant in blood culture specimens, S epidermidis may S epidermidis S saprophyticus cause infection in neonates, the immunocom- promised, and in patients with an indwelling central line, shunt placement, or prosthetic implant. Staphylococcus saprophyticus occurs Streptococcus chiefly in the periurethral and urethral flora Haemolysis where it shows a tropism for urinary tract epi- Beta Alpha (or gamma) thelium and causes urinary tract infections in Bacitracin Optochin Bile esculin sexually active adolescent girls, second only to Escherichia coli in this age group.9 SensitiveResistant Sensitive Resistant Positive Negative Staphylococcus aureus is an important patho- S pyogenes S agalactiae S pneumoniae Viridans Nutrionally variant gen, causing skin infections, osteomyelitis, (most group C, G) group streptococcus pneumonia, and septicaemia. It is dis- 6.5% NaCl growth tinguished on the positive results of coagulase, Positive Negative mannitol fermentation, and deoxyribonuclease Enterococcus S bovis (group D) tests.10 Selective media, such as mannitol salt Growth in tellurite agar, may be used for isolating S aureus when screening for carriage in infection control Positive Negative investigations. In the last decade the prevalence E faecalis E faecium of resistance