| What effect may bacterial contamination have on urine pH? | View Page |
| A patient suspected of a urinary tract infection has a negative nitrite test, but bacteria is present upon microscopic examination. What may have caused a false-negative result? (Choose ALL of the correct answers) | View Page |
| Acid and alkaline urine pH Reasons for acidic urine pH include: A high-meat diet Respiratory/metabolic acidosis Hypochloridemia A high concentration of urine glucose An alkaline pH may be the result of: A vegetarian diet Respiratory/metabolic alkalosis A bacterial infection caused by urease-producing bacteria Urine that contains bacteria can become more alkaline if the specimen remains at room temperature for an extended period of time. A pH can be falsely interpreted as more acidic than it actually is if improper technique is used and excess urine is allowed to pool on the reagent strip. The reagents from the protein pad, that includes an acid buffer, can run over into the pH pad if the strip has these two tests located next to each other. | View Page |
| Urine Specimen The urine specimen should be freshly voided. Urine is an ideal medium for the proliferation of bacteria due to the large amount of urea present. These bacteria metabolize urea, producing ammonia that causes the urine pH to become more alkaline. If there is a delay before performance of the test, the sample should be refrigerated. This will: Prevent urease-producing organisms, such as Proteus and Pseudomonas, from converting urine urea to ammonia, which results in an increased pH. Prevent loss of CO2 which increases pH to the alkaline range. | View Page |
| A urine specimen was collected at 6:00 A.M. and remained at room temperature until it was received in the laboratory at 3:30 P.M. How may the pH of the specimen be affected by the extended time at room temperature if bacteria are present in the specimen? | View Page |
| Bilirubin Characterization Bilirubin, a product of hemoglobin breakdown, is characterized by its yellow pigment. The presence of bilirubin in urine is always abnormal. It is important to note that unconjugated bilirubin cannot be excreted by the kidneys because it is bound to albumin and is not soluble in water. In the liver, bilirubin combines with glucuronic acid through the action of a glucuronyl transferase to form water soluble bilirubin diglucuronide. Under normal circumstances, conjugated bilirubin passes from the bile duct and then to the intestinal tract. Intestinal bacteria reduce conjugated bilirubin to urobilinogen. Approximately half of the urobilinogen is excreted in the feces; most of the other half is recirculated through the liver. A small amount of urobilinogen bypasses the liver and is excreted in the urine. | View Page |
| Nitrite Test The nitrites portion of the reagent strip provides a rapid screening test for the presence of gram-negative bacteria that are often responsible for urinary tract infections. Although urine cultures are still needed to confirm the diagnosis and monitor any urinary tract or kidney infection, the need for a culture may not be obvious because in some cases of early bladder infection, the symptoms may be vague or the patient may be asymptomatic. Diagnosis and treatment of cystitis (bladder infection) is important because if left untreated it may result in kidney damage, impairment of renal function, hypertension and/or septicemia. | View Page |
| Bladder Infections Bladder infections are usually caused by gram-negative bacteria. These bacteria reduce nitrates derived from food to nitrites when urine remains in the bladder three to four hours, sufficient time for this reaction to occur. Nitrite is not present in urine under normal circumstances. When present, nitrites react with p-arsanilic acid to produce a diazonium compound. The diazonium compound in turn couples with 3-hydroxy-1,2,3,4 tetrahydrobenzo-(h)-quinolin to produce a pink color. A first morning, clean, voided midstream specimen is optimal for detecting nitrites in urine. | View Page |
| Test Sensitivity This test is sensitive to 0.06-0.1 mg/dL nitrite ion in urines with a low specific gravity and ascorbic acid concentrations of less than 25 mg/dL. Pink spots or pink edges should not be interpreted as a positive result because some medications can color urine red or turn red in an acid environment. Any degree of uniform pink color should be considered positive, suggesting the presence of 105 organisms/mL. Detection of low levels of nitrite ion may be enhanced by comparing the activated test strip to a white background. It is important to note that color development is NOT proportional to the number of bacteria present. The test is specific for nitrites and does not react with any other substances normally present in urine. Negative results do not necessarily rule out a urinary tract infection because yeasts or gram-positive bacteria unable to reduce nitrites may be the causative agent. | View Page |
| False Negative Results False negative results may occur in urine specimens that did not remain in the bladder a sufficient length of time for the bacteria to reduce a measurable quantity of nitrate to nitrite. Other reasons for false negative results include high specific gravity, ascorbic acid levels above 25mg/dL or low pH (<6). Less frequently, the cause may be due to a lack of sufficient nitrate in the diet (green vegetables) or further reduction of nitrite to nitrogen when large numbers of bacteria are present. In patients receiving antibiotics, the metabolism of the bacteria may be inhibited which would also produce a false negative reaction. | View Page |
| Clinical Significance Early detection of bacteria is important in order to prevent cystitis from developing into inflammation or infection involving the kidney and renal pelvis. The nitrite portion of the test strip can be used to screen individuals who are at risk for developing urinary tract infections, such as diabetics, persons with recurrent infections, or pregnant women. The test is also useful in evaluating the success of antibiotic therapy that is used to treat a bladder infection. | View Page |
| Nitrates in urine are reduce to nitrites by: | View Page |
| Urobilinogen Urobilinogen is a byproduct of hemoglobin breakdown. It is produced in the intestinal tract as a result of the action of bacteria on bilirubin. Almost half of the urobilinogen produced recirculates through the liver and then returns to the intestines through the bile duct. Urobilinogen is then excreted in the feces where it is converted to urobilin. As the urobilinogen circulates in the blood to the liver, a portion of it is diverted to the kidneys and appears as urinary urobilinogen. Up to 1 mg/dL or Ehrlich unit of urobilinogen is present in normal urine. A result of 2.0 mg/dL represents the transition from normal to abnormal and the patient should be evaluated further. It is important to note that the reagent strip cannot determine the absence of urobilinogen. | View Page |
| False Positive Leukocyte Esterase Test A false positive result may occur in the presence of strong oxidizing agents in the collection container. In random urine specimens from women, a positive result for leukocyte esterase may be due to a source external to the urinary tract. Other urine sediment findings such as bacteria, squamous or renal epithelial cells, lymphocytes or red blood cells do not contain esterases, and would not produce a positive leukocyte esterase test. | View Page |
| Middle ear damage in cases of S. pneumoniae infections are caused primarily by: | View Page |
| Review 1 Podschun R. Ullmann U.:
Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors
Clinical Microbiology Reviews. 11(4):589-603, 1998Bacteria belonging to the genus Klebsiella frequently cause human nosocomial infections. In particular, the medically most important Klebsiella species, Klebsiella pneumoniae, accounts for a significant proportion of hospital-acquired urinary tract infections, pneumonia, septicemias, and soft tissue infections.The principal pathogenic reservoirs for transmission of Klebsiella are the gastrointestinal tract and the hands of hospital personnel. Because of their ability to spread rapidly in the hospital environment, these bacteria tend to cause nosocomial outbreaks. Hospital
outbreaks of multidrug-resistant Klebsiella spp., especially those in neonatal wards, are often caused by new types of strains, the so-called extended-spectrum-beta-lactamase (ESBL) producersThe incidence of ESBL-producing strains among clinical Klebsiella isolates has been steadily increasing over the past years. The resulting limitations on the therapeutic options demand new measures for the management of Klebsiella hospital infections.While the different typing methods are useful epidemiological tools for infection control, recent findings about Klebsiella virulence factors have provided new insights into the pathogenic strategies of these bacteria. Klebsiella pathogenicity factors such as capsules or lipopolysaccharides are presently considered to be promising candidates for vaccination efforts that may serve as immunological infection control measures. | View Page |
| Review 2 Citron DM. Appelbaum PC.:
How far should a clinical laboratory go in identifying anaerobic isolates, and who should pay?
Clinical Infectious Diseases. 16 Suppl 4:S435-8, 1993Identification of anaerobic bacteria in specimens from sites of infection due to mixed organisms can be time-consuming and expensive. Laboratories should limit anaerobic workups by testing only those specimens that have been properly collected and transported to the laboratory.Use of selective and differential media for initial processing can provide rapid and relevant information to the clinician. Anaerobes isolated from normally sterile sites and sites of serious infection should always be completely identified. Group-or genus-level identifications may suffice in other instances.The Bacteroides fragilis group of organisms should always be identified because of their virulence and resistance to many antimicrobial agents.Some of the other organisms that warrant identification include Clostridium septicum (associated with gastrointestinal malignancy); Clostridium ramosum, Clostridium innocuum, and Clostridium clostridioforme (which are resistant to antibiotics); Clostridium perfringens (a cause of myonecrosis and gas gangrene,potentially serious infection); anaerobic cocci (which may be resistant to metronidazole and clindamycin); and fusobacteria (which may be virulent and resistant to clindamycin and penicillin). | View Page |
| A gram stain of the serous exudate is shown in the photograph. The appropriate report would read: | View Page |
| Group A Strep A Disk/SXT In follow up to the previous question, the upper image again illustrates the colonies recovered from the blood culture bottle. The colonies are small, transluscent, gray-yellow, and surrounded by a wide zone of beta hemolysis.The size of the colonies compared to the zones of hemolysis suggests a group A streptococcus.The susceptibility to bacitracin (zone of inhibition around the "A" disk)(lower photograph) is virtually diagnostic of a group A streptococcus.The absence of a zone of inhibition around the SXT disk indicates resitance to sulfamethoxazole/ trimethoprim. SXT resistance is also shared by group B streptococci, which are, however, resistant to bacitracin.The resistance to SXT is used for the primary recovery of groups A and B streptococci from specimens with mixed culture. Their resistance allows them to selectively grow out from contaminating bacteria that are inhibited by this antibiotic. | View Page |
| A Brown and Brenn gram stain was performed on one of the tissue biopsy specimens. Organisms were seen as shown in the photograph. Based on the history and the appearance of the bacteria, the most likely identification is: | View Page |
| Review 3 Rouquette C. Berche P. The pathogenesis of infection by Listeria monocytogenes Microbiologia. 12:245-58, 1996 Listeria monocytogenes is a Gram-positive bacterium responsible for severe infections in human and a large variety of animal species. It is a facultative intracellular pathogen which invades macrophages and most tissue cells of infected hosts where it can proliferate. The molecular basis of this intracellular parasitism has been to a large extent elucidated. The virulence factors, including internalin, listeriolysin O, phospholipases and a bacterial surface protein, ActA, are encoded by chromosomal genes organized in operons. Following internalisation into host cells, the bacteria escape from the phagosomal compartment and enter the cytoplasm. They then spread from cell to cell by a process involving actin polymerisation. In infected hosts, the bacteria cross the intestinal wall at Peyer's patches to invade the mesenteric lymph nodes and the blood. The main target organ is the liver, where the bacteria multiply inside hepatocytes. Early recruitment of polymorphonuclear cells lead to hepatocyte lysis, and thereby bacterial release This causes prolonged septicaemia, particularly in immunocompromised hosts, thus exposing the placenta and brain to infection. The prognosis of listeriosis depends on the severity of meningoencephalitis, due to the elective location of foci of infection in the brain stem (rhombencephalitis). Despite bactericidal antibiotic therapy, the overall mortality is still high (25 to 30%). | View Page |
| Clostridium difficile Another organism that has more recently become problematic is Clostridium difficile. Usually, normal gut flora resist overgrowth and colonization by this organism. However, antibiotic use that suppresses the normal gut flora, allows proliferation of C. difficile. The organism releases toxins that cause inflammation and damage to the mucosal lining of the colon, leading to severe diarrhea. An antibiotic-resistant strain has developed that can result in colitis, sepsis, and death. Elderly patients, patients with severe underlying illness, and patients undergoing immunosuppressive therapy are at higher risk of becoming infected since their immune response to the bacteria and its toxins is diminished. | View Page |
| Screening cultures for MRSA Surveillance is a critical component of any program for controlling multi-drug resistant organisms. Many institutions are using active surveillance cultures to identify patients who are colonized with a targeted MDRO. With respect to MRSA, an increasing number of hospitals are screening patients upon admission and on a periodic basis (usually weekly). The anterior nares is the primary site that is swabbed for screening.There are several selective and/or differential media that can be used for this purpose.Baird Parker Agar is a selective medium for the isolation of S. aureus; on this medium S. aureus produces black colonies with a clear halo.Mannitol Salt Agar is also a selective medium; S. aureus produces yellow colonies which contrast with the red color of the medium.Chromogenic agars have been developed for the isolation and presumptive identification of different species of bacteria and yeast. The media are formulated so that as different organisms utilize various substrates in the media, the organism of interest produce colonies with a unique color. Chromogenic agars specifically designed for the detection of MRSA are commercially available.In addition to culture methods, there are now commercially available, FDA approved methodologies for screening for MRSA by PCR. Although equipment and cost factors may not make these a viable option for every laboratory, they may offer greater sensitivity and improved turnaround times. | View Page |
| Newer antibiotics for Treatment of Resistant Enterococci Synercid (quinupristin and dalfopristin) is an alternative therapy for systemic infections with VRE. It is FDA approved for treatment of life threatening infections with vancomycin resistant isolates of E. faecium. This antibiotic is appropriately suppressed from reports of other vancomycin sensitive strains of E. faecium, and all other species of Enterococcus.Linezolid belongs to the oxazolidinone class of antibiotics. It is active against VRE. The main indication of linezolid is treatment of severe infections caused by gram positive bacteria that are resistant to other antibiotics.Daptomycin is the first lipopeptide released onto the market. Daptomycin has been approved for the treatment of skin and soft tissue infections; evaluaton of efficacy in more serious systemic infections is ongoing. Disk diffusion testing for Daptomycin is not reliable.Telavancin is a lipoglycopeptide under trial for MRSA and other gram positive infections. The spectrum of activity of telavancin is similar to vancomycin, but it may be active against some VRE strains (Van B). | View Page |
| Blood culture bottles Are used to collect sterile blood samples from patients who may be septic (have bacteria or other organisms growing in their bloodstream).
Different blood culture bottles are used for aerobic, anaerobic, and pediatric collections. | View Page |
| Introduction Blood is normally sterile. Any bacterial growth in the bloodstream is abnormal, and is an important cause of fever.Blood culture means the incubation
of blood in appropriate media to allow growth and identification of bacteria or other organisms that may be present in a patient’s bloodstream.
Blood cultures are performed on febrile patients to identify and treat bloodborne organisms with the most appropriate antibiotic. | View Page |
| Site preparation continued Finally, prep the site with an iodine swab.
Start at the center of the site, and move outward in enlarging concentric circles. Do not go over the same area twice.Stop when you have covered an area about 4 inches in diameter. Allow this area to dry for at least one minute so that the iodine has time to kill the skin bacteria. | View Page |
| Avoid skin contamination Normal skin is not sterile – it contains numerous bacteria.These normal skin bacteria can contaminate a blood culture, causing a false-positive blood culture result.Thorough decontamination of the skin puncture site is therefore essential prior to obtaining the blood culture specimen. | View Page |
| Site preparation Clean the site thoroughly with alcohol, then with iodine, to rid the skin of contaminating bacteria.Next, clean the site again with alcohol, and allow it to dry.
| View Page |
| 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. | View Page |
| 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. | View Page |
| Cerebrospinal Fluid and Specimens Collected from Other Sterile Sites Cerebrospinal fluid (CSF) and all specimens collected from sterile sites should have a microscopic examination performed along with culture. Bacteria found in CSF, blood, tissue, and specimens from other sterile sites are always significant.CSF should be cytospun, if possible, to increase the chance of detecting a pathogen. The quantity of organisms seen and the amount and type of host cells, e.g., mononuclear or polymorphonuclear (PMN) white blood cells, is important to report. The presence of PMNs indicates bacterial infection. It is also important to determine and report whether the bacteria are found inside or outside of white blood cells. | View Page |
| 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.” | View Page |
| 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. | View Page |
| A large number of which of these types of cells would indicate that a sputum specimen is sub-optimal and should not be used for culture? | 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. | View Page |
| Gram-positive Cocci 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 | View Page |
| Intracellular Bacteria Sometimes bacteria are present within white cells. It is important to note on the report whether the bacteria are intracellular. In this field, gram-negative bacteria are present within a neutrophil as indicated by the arrow. | View Page |
| What are the structures indicated by the arrows in this field from a Gram-stained smear? | View Page |
| Gram-positive Bacilli Bacilli are rod-shaped bacteria. Gram-positive bacilli are clearly visible in this Gram stain field. | View Page |
| Gram-negative Bacilli Most bacteria that are encountered in the clinical microbiology laboratory will be gram-negative bacilli. Gram-negative bacilli include the Enterobacteriaceae, nonfermentative bacilli, Haemophilus species, and several fastidious species. Gram-negative bacilli can be observed in this field. | View Page |
| The bacteria in this slide are gram-negative cocci. | View Page |
| Significance of Specific Findings When evaluating Gram stains of clinical samples, keep in mind the source of material from which the smear was made. Bacteria found in cerebrospinal fluid (CSF), blood, tissue and specimens from other sterile sites are always significant. Gram stains of body fluids that are normally sterile must be examined carefully. For every one organism per oil immersion field, there are about 105 organisms per mL present in the sample! Examining stained smears of CSF sediment may assist the clinician in establishing a presumptive diagnosis. The Gram stain result and the results of other special stains could also guide in the selection of culture media. If bacteria are observed in a CSF specimen, it is important to determine and report whether the bacteria are inside or outside of white blood cells (intracellular or extracellular). The quantity of organisms seen and the amount and type of host cells are also important to report. Bacteria observed in specimens from the throat, genital tract and other areas containing normal flora suggest infection only if their composition and type varies significantly from the norm. | 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) | View Page |
| Gram Stain Principle Bacteria that retain the primary crystal violet stain that is first applied as part of the staining process are classified as gram-positive bacteria. The stain is retained because the cell wall of a gram-positive bacteria is not as permeable as the cell wall of a gram-negative bacteria. Therefore, when the decolorizer is applied to the smear, the crystal violet will be retained if the bacteria is gram-positive and the bacteria will appear blue or purple. Prior to adding the decolorizer, iodine is applied to the smear. The iodine fixes the crystal violet stain to the cellular components of gram-positive bacteria, further preventing the decolorization of the gram-positive organisms. The bacteria indicated by the arrows in Image 1 are gram-positive bacteria.Image 1If the bacteria is gram-negative, decolorizer will pull the crystal violet stain out of the cell walls, allowing the bacteria to be stained with the counterstain so that a gram-negative bacteria appears red or pink. The arrows in Image 2 point to gram-negative bacteria.Image 2 | View Page |
| What is the purpose of the iodine that is used in the Gram stain procedure? | View Page |
| Under-decolorized or Over-decolorized Smears Although this smear is the proper thickness, it is not appropriate for examination because the host cells are stained blue instead of red, indicating that the smear was under-decolorized. In addition, small flecks of precipitated stain are present. Notice that the precipitated stain is irregular in shape, which helps differentiate the flecks from bacteria. A slide is also not acceptable for examination if microorganisms that should be gram-positive appear pink. This may indicate that either the Gram's iodine was not applied or the slide was over-decolorized. Staining gram-positive and gram-negative control slides along with the patient's smear would confirm that proper staining technique was used. If it is impossible to prepare a new smear, the poorly stained smear may still be salvaged. Remove immersion oil from the smear using xylol. Use appropriate procedures and personal protective equipment when using xylol, since it is a hazardous chemical. If the smear is under-decolorized, repeat the decolorization and counterstain steps. If the smear is over-decolorized, the slide should be stained again. | View Page |
| Which of the following statements is true regarding the Gram-stained smear that is represented by this image? | View Page |
| In a properly decolorized area, epithelial cells, red blood cells, white blood cells and most background material will stain pink or red. It is in this area that the gram stain reaction of the bacteria can be correctly interpreted. | View Page |
| Under-decolorized smear Although this smear is the proper thickness, it is not appropriate for examination because the nonbacterial elements are stained blue instead of red. In addition, small flecks of precipitated stain are present. Notice that the precipitated stain is irregular in shape, which helps differentiate the flecks from bacteria. The stain should have been filtered before using. | View Page |
| Identification of bacteria Identification of bacteria in direct smears may be of lifesaving importance. For example, 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. The appearance of bacteria on gram-stained smears is suggestive of a certain species, but identification may not be made on the basis of the stain alone. An exception to this rule is the presence of gram-negative intracellular diplococci from a male urogenital specimen, which is presumptive identification of Neisseria gonorrhoeae. In addition, culture results can be correlated with the direct smear report. | View Page |
| Intracellular bacteria. Sometimes bacteria are present within white cells. In this field, there are gram negative bacteria present within a neutrophil.
| View Page |
| Principle Gram stained direct smears are examined using the oil immersion objective (100x) of the microscope. The quantity and type of bacteria and nonbacterial cellular elements present is recorded. | View Page |
| Size and Appearance of Cellular Elements Type of Cell Size Shape Stain Epithelial cells 25 microns irregular pink/red White blood cells 12 microns round pink/red Red blood cells 7 microns round pink/red Yeast 7 microns ovoid blue Bacteria 0.5 - 1 micron variable blue or red Epithelial cells are larger than white blood cells and red blood cells, and contain a single nucleus. White blood cells (neutrophils contained in pus) usually show a segmented nucleus. Red blood cells are 1/2 to 2/3 as large as white blood cells, contain no nucleus, and are Gram negative. Hyphae are gram positive tubular filamentous fungal elements which may show branching or intertwining. Yeast cells are round to oval, often budding, Gram positive fungal elements, about the same size as RBCs. They are generally much larger than bacteria. | View Page |
| Recording Bacterial Characteristics The Gram stain reaction, shape, and arrangement of bacteria, and the presence or absence of intracellular organisms must be noted on the worksheet.Examples:Gram positive cocci in chains are present.Gram negative diplococci, intracellular, are present within white blood cells.Quantitate by approximating the average number of each cell type seen in 10 oil immersion fields, and record as:Many = More than 15/fieldModerate = 4-15/fieldFew = 1-3/fieldOccasional = 2-10/10 fieldsRare = 0-2/10 fields | View Page |
| Significance of Specific Findings: Epithelial cells in large numbers within sputum smears means that the specimen is predominantly oral saliva, rather than true sputum from the lung. Epithelial cells in urine smears indicate that the sample has been contaminated by organisms found on the vulva or distal urethra. Bacteria found near or on epithelial cells are usually normal contaminating bacterial flora.White blood cells indicate inflammation and possible infection. The direct smear examination should focus within and around these cells.Red blood cells in a direct smear are not usually significant.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.Hyphae are more likely to indicate the presence of fungal infection, but this determination requires correlation with clinical findings.Bacteria found in spinal fluid, blood, tissue and specimens from other sterile sites are always significant.Body fluids which are normally sterile must be examined carefully. If only one organism per oil immersion field is identified, then there are about 105 organisms per mL present in the sample! Bacteria observed in specimens from the throat, genital tract and other areas containing normal flora suggest infection only if their composition and type varies significantly from the norm. | View Page |
| The ends of rod-shaped bacteria may appear: | View Page |
| A mixture of Gram positive and Gram negative bacteria may be present within the same field of a slide. | View Page |
| Cocci and Rods Bacteria may be either round (cocci) or rod-shaped (bacilli). Either shape may be Gram positive or Gram negative. A mixture of Gram positive and Gram negative bacteria can occur in the same field. Examples of Gram positive bacilli (rods) are shown on the bottom left, and Gram positive cocci are shown on the bottom right. | View Page |
| Chain Cell Arrangement The long chains of Gram positive bacteria are obvious in this field. | View Page |
| Clusters of Gram Positive Bacteria Clumps or clusters of Gram positive bacteria are evident in this slide. | View Page |
| Focus Power Use the lower power objective to focus on the field, and then switch to the 100x oil immersion objective to examine the appearance of the bacteria present. | View Page |
| Pair Cell Arrangement This field shows examples of Gram positive bacteria in pairs. These are lancet shaped "diplococci" which could represent Streptococcus pneumoniae. However, determination of bacterial species always requires confirmatory testing. This particular image is not from a culture, but from a Gram stained sputum specimen. The large Gram negative pink staining rounded structures in the background are neutrophils. | View Page |
| Gram Negative Bacilli Examples of Gram negative bacilli or rod shaped bacteria are present in this field. | View Page |
| Gram Stain The Gram stain provides a fundamental differentiation between types of bacteria. Gram positive bacteria retain the primary stain and are deep violet in color. Gram negative organisms are those that decolorize during the staining process and, after counterstaining, are pink in color. Bacterial organisms occur in two basic shapes: cocci, which are spherical, and bacilli, (also known as rods), which are elongated. Both cocci and rods may be either Gram positive or Gram negative. Examples of Gram positive cocci are shown in the upper image on the right and Gram negative cocci are shown in the lower image. | View Page |
| Single Cell Arrangement These Gram negative bacteria are in a single cell arrangement.
| View Page |
| Tetrad Cell Arrangement Examples of Gram positive bacteria, many appearing as tetrads, are seen in this slide. Sometimes bacteria in tetrad may not be as clear cut, but may appear as clusters. N. gonorrhea colonies may appear as Gram negative tetrads, especially if the smear is made from very young colonies. | View Page |
| The bacteria on both the positive and the negative control slides are observed to stain pink. This finding will not affect the rest of the slides in the batch. | View Page |
| The sediment from a freshly collected urine specimen is examined microscopically. In addition to bacteria, what other finding from the list below would further indicate the presence of a urinary tract infection (UTI)? | View Page |
| The patient was a female and the urinalysis was completed within two hours of collection. Which of the following findings are inconsistent with the rest of the report? | View Page |
| The patient is a female and the urinalysis is completed within two hours of collection.True or false? The presence of bacteria correlates with the slight turbidity and WBC result. | View Page |
| What are the microscopic elements that are indicated by arrows in this image? | View Page |
| Specimen Collection and Storage Examination of a fresh urine specimen provides the best results. If a specimen cannot be examined immediately, it may be refrigerated for up to 12 hours. Refrigeration prevents decomposition of casts, cells and the overgrowth of bacteria. The urine sediment elements begin to lyse after 1-3 hours at room temperature.
Although the most commonly received urine specimen is the random urine collection, the specimen of choice for urinalysis is the first morning urine. The first morning urine is more concentrated and allows for the detection of substances which may not be present in a more dilute random sample.
Once the physical and chemical characteristics of the urine have been determined, the microscopic exam is performed on the sediment. | View Page |
| Bacteria Under Phase Contrast Under phase contrast, the bacteria appear very dark. The larger cells are white blood cells (blue arrow). | View Page |
| Bacteria Bacteria may also be present, especially during a urinary tract infection. This view shows bacteria as solid gray rods or cocci. Since bacteria may also be a contaminant in specimens remaining at room temperature, or due to an unclean catch, caution must be observed in reporting bacteria. If 20 organisms per hpf are seen, the bacteria are considered to be clinically significant. | View Page |
| Specimen #4 - Adult Male The results of this specimen are abnormal but the abnormalities correlate with each other. The turbidity can be explained by the presence of bacteria and crystals. The presence of RBCs in the microscopic explains the blood found on the dipstick. The casts, bacteria and WBCs can account for the increased protein. The results may be reported. | View Page |
| Specimen #3 - Adult Female The results are abnormal. The presence of glucose is not a normal finding. However, the two glucose methods correlate well with each other.The specific gravity does not correlate well with the glucose. A large amount of glucose should elevate the urine specific gravity. The specific gravity result should therefore be rechecked before reporting. The presence of 3+ bacteria, does not correlate well with scant white cells and lack of turbidity. The technologist should question whether the specimen was held at room temperature for a protracted period prior to examination. | View Page |
| Estimating Elements The number of bacteria, yeast, crystals and mucous must be estimated. Examine 10 fields under high power (40X) magnification. Use phase-contrast if needed. Determine the average of each element found and record the findings according to the following criteria: Element fills <1/4 of each field = trace Element fills about 1/4 of each field = 1+ Element fills about 1/2 of each field = 2+ Element fills about 3/4 of each field = 3+ Element fills total field = 4+ | View Page |
| Bacteria and Amorphous Material A mixture of bacteria and amorphous material is seen in this field. In order to determine the approximate number of bacteria, the amorphous material can be eliminated. | View Page |
| A urine specimen was delayed in transport to a medical laboratory. Upon examination of the urine sediment, the technologist cannot distinguish between bacteria and amorphous urates. Which of the following reagents would dissolve the crystals? | View Page |
| Iodine will confirm the presence of: | View Page |
| Amorphous Urates Amorphous urates can be dissolved in dilute sodium hydroxide. Amorphous phosphates will dissolve in dilute acetic acid. In either case, the bacteria will remain. | View Page |
| Variations in Morphology Many variations in morphology may be seen when examining Wright's stained peripheral blood smears. One method of classifying these variations in white cell morphology is based on the way the body responds to a stimulus, deficiency, or the presence of an inherited defect. This classification falls into three groups:Pathological:
Cells may show abnormalities in appearance and/or function. The body is responding abnormally to a stimulus or inherited defect, resulting in physiological impairment in the patient.
Nonpathological:
Cells may show variation in morphology but their function is normal. Their presence does not cause physiological impairment.
Reactive:
Cells show variation in morphology but are functioning normally in response to a specific stimulus, such as a virus or bacteria. There is a disease process in progress to which the cells are responding. Although the morphology has varied from normal and their presence is significant, the body is responding normally to a stimulus. | View Page |
| Match the following: | View Page |
| Unusually Darkly Staining Dohle Bodies Unusually dark staining Dohle bodies are seen in the cytoplasm of this cell, along with bacteria, an infrequent finding. The bacteria are indicated by the red arrows and stain almost black. The Dohle bodies are patches of dark blue stained material, indicated by the blue arrows. | View Page |
| Classification Vacuoles, toxic granulation and degranulation are classified as reactive since the body is responding normally in an effort to rid itself of infection caused by bacteria. Morphological changes related to aging are also classified as reactive. | View Page |
| Which morphologic term describes this slide? | View Page |
| Which morphologic term describes this slide? | View Page |
| Which morphologic term describes this slide? | View Page |
| Which morphologic term describes this slide? | View Page |
| Chediak-Higashi Chediak-Higashi syndrome is a rare autosomal recessive disorder. It results from a mutation of the gene LYST which encodes a protein with multiple phosphorylation sites. This defect causes a cellular abnormality involving the fusion of cytoplasmic granules. Early in neutrophil maturation normal azurophilic granules form, but they fuse together to form megagranules. Later during the myelocyte stage, normal specific granules form. The mature neutrophils contain both normal specific granules and abnormal azurophilic granules.
These large abnormal granules can be seen in the cytoplasm of neutrophils, eosinophils, basophils, monocytes and lymphocytes.
These abnormal granules are able to kill bacteria in neutrophils and monocytes; however, the process is much less effective than in normal cells in part, because these neutrophils have impaired locomotion. For these reasons, individuals with Chediak-Higashi have recurrent infections.
An accelerated lymphoma-like phase occurs, with lymphadenopathy, hepatosplenomegaly, and pancytopenia. Death often occurs at an early age.
| View Page |
| Which morphologic term describes this slide? | View Page |
| Which morphologic term describes this slide? | View Page |