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Organism Information and Courses from MediaLab, Inc.

These are the MediaLab courses that cover Organism and links to relevant pages within the course.

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Case Studies in Clinical Microbiology
A 25-year-old female presented in the emergency room with an acute urethral discharge of two days duration. The image to the right shows the Gram stained smear that was obtained. Many polymorphonuclear leukocytes and intracellular and extracellular gram-negative diplococci were observed. Based on the clinical history and the Gram stain observation, a diagnosis of gonorrhea can be made.View Page
The positive oxidase reaction as shown in the image (yellow arrow) rules out which of the following two look-alike organisms of N. gonorrhoeae? (Choose all that apply)View Page
Extended-Spectrum Beta-Lactamase (ESBL) Activity

Illustrated in the image is the surface of a disk diffusion test including a 30 mg ceftazidime disk (left) and a combintation 30/10 mg ceftazidime/clavulanic acid disk (right). Observe in the photograph that the zone of inhibition around the the combination ceftazidime/clavulanic acid disk (right) is at least 5 mm larger than around the clavulanic acid disk (left). This observation that the presence of clavulanic acid, a beta-lactamase inhibitor, has resulted in such a large increase in the zone of inhibition indicates that an extended-spectrum beta-lactamase (ESBL)is being produced. Additionally, the Clinical and Laboratory Standards Institute (CLSI) Performance Standards for Antimicrobial Testing Standards, published January 2011, proposes in the M100-S21document, table 2A-S1, that cefotaxime (30 mg) and cefotazime-clavulanic acid (30/10 mg) testing per performed alone AND in combination with the ceftazidime and ceftazidime/clavulanic acid testing previously described. When an organism is producing an ESBL, the susceptibility to individual cephalosporins cannot be predicted, thus requiring that each drug must be tested individually.

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Review 1

Garbutt JM. Littenberg B. Evanoff BA. Sahm D. Mundy LM. Enteric carriage of vancomycin-resistant Enterococcus faecium in patients tested for Clostridium difficile. Infection Control & Hospital Epidemiology. 20(10):664-70, 1999 OBJECTIVE: To identify independent risk factors for enteric carriage of vancomycin-resistant Enterococcus faecium (VREF) in hospitalized patients tested for Clostridium difficile toxin. PATIENTS: Convenience sample of 215 adult inpatients who had stool tested for C. difficile between January 29 and February 25, 1996. RESULTS: 41 (19%) of 215 patients had enteric carriage of VREF. Five independent risk factors for enteric VREF were identified: (1) history of prior C. difficile infection, (2) parenteral treatment with vancomycin for > or = 5 days, (3) treatment with antimicrobials effective against gram-negative organisms, (4) admission from another institution, and (5) age > 60 years. These risk factors for enteric VREF were independent of the patient's current C. difficile status. CONCLUSIONS: Antimicrobial exposures are the most important modifiable independent risk factors for enteric carriage of VREF in hospitalized patients tested for C. difficile.

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Review 2

Suppola JP. Kuikka A. Vaara M. Valtonen VV. Comparison of risk factors and outcome in patients with Enterococcus faecalis vs Enterococcus faecium bacteremia. Scandinavian Journal of Infectious Diseases. 30(2):153-7, 1998. The purpose of our study was to determine retrospectively the risk factors for the acquisition of Enterococcus faecalis vs E. faecium bacteremia, as well as the clinical outcomes of these patients. 62 patients with Enterococcus faecalis bacteremia were compared to 31 patients with E. faecium bacteremia. Haematologic malignancies, neutropenia, high-risk source and previous use of aminoglycosides, carbapenems, cephalosporins and clindamycin were significantly associated with E. faecium bacteremia. Instead, urinary catheterization was found to be related to Enterococcus faecalis bacteremia. The mortality rates within 7 d and 30 d were 13% and 27%, respectively, in patients with E. faecalis bacteremia and 6% and 29%, respectively, in patients with E. faecium bacteremia. There was no difference in mortality between E. faecalis and E. faecium bacteremia, nor was there a difference in seriousness of disease at the time of bacteremia. In the subgroups of patients with monomicrobial or clinically significant E. faecalis vs E. faecium bacteremia, the mortality rates were similar to the results of all subjects. Our results do not support the theory that E. faecium would be a more virulent organism than E. faecalis.

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What is the most important modifiable risk factor for enteric colonization with vancomycin-resistant Enterococcus faecium (VREF)?View Page
Review 1

Lorimer JW. Eidus LB.: Invasive Clostridium septicum infection in association with colorectal carcinoma. Canadian Journal of Surgery. 37:245-9, 1994 The association between invasive Clostridium septicum infection and colorectal carcinoma is examined by the presentation of three cases and a review of the literature. In the first two cases the patients presented with nontraumatic metastatic clostridial gas gangrene. In the third case a patient with chemotherapy-induced myelosuppression from concomitant multiple myeloma had a necrotizing transmural infection of the right colon. The apparent portal of entry of Clostridium septicum was an occult carcinoma of the ascending colon. The increasing evidence for a strong link between this organism and some cases of neutropenic enterocolitis is reviewed.

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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, 1993 Identification 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).

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A clinical condition often associated with Streptococcus anginosus ("milleri") is:View Page
Review 1

Piscitelli SC., Shwed J., Schreckenberger P., Danziger LH. Streptococcus milleri group: renewed interest in an elusive pathogen. European Journal of Clinical Microbiology & Infectious Diseases.11:491-8, 1992 The following review examines the bacteriological characteristics, epidemiology, pathogenicity and antimicrobial susceptibility of the "Streptococcus milleri group". "Streptococcus milleri group" is a term for a large group of streptococci which includes Streptococcus intermedius, Streptococcus constellatus, and Streptococcus anginosus. Usually considered commensals, these organisms are often associated with various pyogenic infections including cardiac, intra-abdominal, subcutaneous and central nervous system infections, particularly with the formation of abscesses. Organisms of the "Streptococcus milleri group" are often unrecognized pathogens due to the lack of uniformity in classifications and difficulties in microbiological identification. Penicillin G, cephalosporins, clindamycin and vancomycin all possess activity against these streptococci. Use of agents with poor activity may promote infections with "Streptococcus milleri group" and allow it to exhibit its pathogenicity. An understanding of these organisms may aid in their recognition and proper treatment.

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Review 1

Spencer RC.: Invasive streptococc European Journal of Clinical Microbiology & Infectious Diseases. 14 Suppl. 1:S26-32, 1995. Before the introduction of antibiotics, serious infections caused by Streptococcus pyogenes (Lancefield Group A streptococci) were common. Before World War II, this bacterium was responsible for as many as 50% of postpartum deaths and was the major cause of death in patients with burns. Also common were the sequelae of streptococcal infections-rheumatic fever and post-streptococcal glomerulonephritis. With the use of penicillin, however, Streptococcus pyogenes was believed to be virtually eliminated as a pathogen. The organism was consigned to the history books, but not for long. In the mid-1980s, focal resurgences of rheumatic fever began to be reported from different areas in the USA, such as Salt Lake City, Utah. In such communities, where increases in cases of rheumatic fever had been reported, the serotypes M-1, 3, 5, 6 and 18 were isolated which, on culture, produced characteristic mucoid colonies. At the same time, reports of increases in invasive streptococcal disease began to surface in both the US and Europe. Two syndromes were described; invasive streptococcal infection, occurring in previously healthy children and adults, commonly associated with septicaemia resulting from a deep focus of infection such as bone or lung; and streptococcal toxic shock syndrome, involving a cutaneous focus, accompanied by necrotizing or bullous soft tissue changes. Septicaemia is rare in streptococcal toxic shock syndrome, but the most characteristic feature is one of rapidly progressing multi-organ failure. A high proportion of the strains of Streptococcus pyogenes associated with this condition are serotype M-1, and fatality rates approaching 50% have been reported.

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Clinical History

The prototype history for this organism is either a still birth or a neonate with death ensuing within two or three days post-partum due to high fever, sepsis, and respiratory distress. The mother usually experienced a flu-like illness late in the third trimester of pregnancy, characterized by low-grade fever, myalgias, malaise, and backache. In this case, biopsy material of brain tissue obtained at autopsy was submitted to the pathology laboratory for tissue diagnosis and fluid from the pia-arachnoid was sent to the microbiology laboratory for culture.

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A Brown and Brenn gram stain was performed on one of the tissue biopsy specimens. Organisms were seen as shown in the image. Based on the history and the appearance of the bacteria, the most likely identification is:View Page
Listeria Gram stain

Image of a Gram stain prepared from an 18 hour old broth culture. The organism is a small, non-spore-forming bacillus measuring between 0.4 to 0.5um by 1.0 to 3.5um. Coccobacillary forms, diplobacilli, and bacilli in short chains or in diphtheroidal clusters (upper right in image) may all be observed in Gram stain preparations. When found in the CSF, the organism may be found both intracellularly and extracellulary.

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Which of the following is NOT related to the virulence of Listeria monocytogenes? View Page
Which of the following factors has NOT led to the current increase in incidence of listeriosis?View Page
In view of the feedback to the previous question, what is the most likely reason that the clinical correlation does not seem to fit in this case?View Page
Review 2

Griego RD. Rosen T. Orengo IF. Wolf JE.: Dog, cat, and human bites: a review. Journal of the American Academy of Dermatology. 33:1019-29, 1995 It is estimated that half of all Americans will be bitten by an animal or another human being during their lifetimes. The vast majority of the estimated 2 million annual mammalian bite wounds are minor, and the victims never seek medical attention. Nonetheless, bite wounds account for approximately 1% of all emergency department visits and more than $30 million in annual health care costs. Infection is the most common bite-associated complication; the relative risk is determined by the species of the inflicting animal, bite location, host factors, and local wound care. Most infections caused by mammalian bites are polymicrobial, with mixed aerobic and anaerobic species. The clinical presentation and appropriate treatment of infected bite wounds vary according to the causative organisms. Human bite wounds have long had a bad reputation for severe infection and frequent complication. However, recent data demonstrate that human bites occurring anywhere other than the hand present no more of a risk for infection than any other type of mammalian bite. The increased incidence of serious infections and complications associated with human bites to the hand warrants their consideration and management in three different categories: occlusional/simple, clenched fist injuries, and occlusional bites to the hand. This article reviews dogs, cat, and human bite wounds, risk factors for complications, evaluation components, bacteriology, antimicrobial susceptibility patterns, and recommended treatments. Epidemiology, clinical presentation, and treatment of infections caused by Pasteurella multocida, Capnocytophaga canimorsus, Eikenella corrodens, and rhabdovirus (rabies only) receive particular emphasis.

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Cerebrospinal Fluid (retired 7/17/2012)
Specimen Handling and Storage

The stability of the CSF sample varies depending on the procedures ordered. Cell counts are ALWAYS STAT and should be performed within 30 - 60 minutes for best results. Samples should be left at room temperature for no longer than one hour and refrigerated following testing. Refrigeration is not recommended for culture specimens since fastidious organisms such as Haemophilus influenzae and Neisseria meningitidis may not survive the cold temperature.

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Chemical Screening of Urine by Reagent Strip (retired March 2012)
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.

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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.

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Fundamentals of Molecular Diagnostics (retired 2/12/2013)
Why Choose a Molecular Method?

A molecular method may be the test method of choice for a variety of reasons, including the following:Organisms of interest, especially in microbiology or virology, may be extremely slow growing, fastidious, or extremely limited in quantityOther technologies lack the necessary sensitivity or specificityPatient population requirements

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General Laboratory Question Bank - Review Mode (no CE)
What is the purpose of using acetone/alcohol in the Gram stain procedure?View Page
A definitive diagnosis of malaria can be made by:View Page

HIPAA Privacy and Security Rules
Case Study: De-identified Health Information. You work in a laboratory microbiology department that provides a local nursing home with information about the effectiveness of various antibiotics it uses to treat infections. You print the requested information, including patient first and last names, birthdates, and medical record numbers, bacterial organisms identified, and the organisms' sensitivities to various antibiotics. What information should you provide to the nursing home?View Page

Histology Special Stains: Carbohydrates
Mucicarmine: Diagnostic Applications

The mucicarmine staining procedure is very specific in its detection of mucins of epithelial origin and is used to identify adenocarcinomas. It is particularly useful in detecting adenocarcinomas originating in the gastrointestinal tract. This procedure can also successfully stain and detect the capsule of a fungal organism known as Cryptococcus neoformans . This fungus is usually identified in the lungs and in nervous tissues. C. neoformans is more likely to infect immunocompromised patients than persons with active immune systems.

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Human Papillomavirus (HPV) and Molecular Diagnostic Testing
Molecular Testing Basics

Molecular diagnostics or molecular tests are assays that detect nucleic acids, DNA or RNA, in a sample. It is usually impossible to detect a whole strand of DNA or RNA; most often a specific region of DNA or RNA (called a target) that is unique to the organism, mutation, or disease is detected. Because these assays are detecting or quantifying biomolecules that are minute in size and concentration, special techniques are employed. Most assays include a component that is labeled and detection or measurement of the label is used to determine the presence or quantity of target in each sample.Two common techniques used in molecular testing are hybridization and amplification.

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Introduction to Bioterrorism
Biological Agents

Biological agents are organisms or toxins that can kill or incapacitate people, live stock, and crops. The three basic groups of biological agents that would likely be used as weapons are bacteria, viruses, and toxins. Biological agents can be dispersed as aerosols or airborne particles.

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Agent: Pneumonic plague (Bacterium)

Most likely means of dissemination: AerosolPrimary route of entry: InhalationGeneral signs and symptoms: High fever, chills, headache, coughing up of blood (hemoptysis), and toxemia, progressing rapidly to difficulty in breathing (dyspnea), and bluish discoloration of the skin and mucous membranes (cyanosis).There is another form of the disease called “bubonic plague”. While it is caused by the same organism, it is not transmissible through human contact. Pneumonic plague is transmissible through human contact.

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Laws and Rules of the Florida Board of Clinical Laboratory Personnel (retired 9/1/2010)
Description of Specialties (1)

Specialists in microbiology perform testing to diagnose and stop the spread of infectious organisms, including bacteria, viruses, and parasites. Specialists should be able to isolate and identify a wide variety of these organisms. Testing procedures include direction examination and antigen detection methods. Specialists in serology and immunology measure antibodies to infectious organisms. Specialists should be familiar with all serology techniques (except those specific to immunohematology). This specialty includes all lab procedures performed in the specialty of histocompatibility. Specialists in hematology must be able to identify and evaluate cells in blood and bone marrow and identify disorders of these cell. Specialists should be familiar with routine and special tests to determine the number, morphology, and function of cells in body fluid.

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Microbiology / Serology Question Bank - Review Mode (no CE)
This suspicious form, shown below at both low (10X) and high dry (40X) power, measures 90 micro meters by 42 micro meters. It was seen in a stool sample.View Page
This parasite is found in blood.View Page
This suspicious form, found in urine, measures 120 micro meters by 50 micro meters.View Page
I reside inside red blood cells, where I grow and grow until the cells are eventually destroyed.View Page
Which of the following statements about Rickettsia is false:View Page
Which of the following organisms is the most common cause of acute cystitis:View Page
The capsular material used to identify capsular subtypes of Pneumococci consists of:View Page
The most frequent cause of bacterial meningitis in older adults is:View Page
Which of the following organisms in most frequently associated with endocarditis:View Page
Which one of the following statements about Coxiella burnetii is not true:View Page
Match the Streptococcal organism with the appropriate group.View Page
Which of the following organisms will give a positive coagulase test:View Page
Which of the following organisms is an obligate intracellular parasite:View Page
VDRL is an example of which of the following types of tests:View Page
The Quelling test is useful for which of the following :View Page
Which of the following is used as the indicator in the rapid carbohydrate utilization tests:View Page
Which two of the following organisms are gram positive:View Page
Which of the following organisms are gram negative:View Page
Which of the following organisms are gram positive:View Page
Which of the following organisms are gram positive:View Page
Which of the following substances produced by Group A Streptococci is responsible for producing type specific immunity:View Page
Gram positive organismsView Page
The slide coagulase test is a rapid method for identifying which of the following organisms.View Page
Which one of the following organisms is typically urease negative:View Page
The oxidase test is used to presumptively identify:View Page
Which one of the following tests would be positive in the presence of Klebsiella:View Page
Which of the following organisms is best visualized by use of a darkfield microscope:View Page
With regard to blood cultures, which blood to broth ratio is most conducive to growth:View Page
On sheep blood agar Haemophilus influenzae may exhibit satellite formation around all but which of the following organisms:View Page
Which one of the following organisms do not usually stain with an acid-fast stain, or one of its variants:View Page
Match type of media on the right with best description:View Page
Which of the following organisms is not an aerobic organism:View Page
Which of the following tests would be used to directly document the presence of a specific organism in a clinical specimen:View Page
MacConkey agar contains all of the following except :View Page
Which of the following specimens would not be considered suitable for anaerobic culture:View Page
Match the organisms on the right with culture medium:View Page
Match the culture media on the right with possible organism on the left:View Page
Match the organism on the right with the most suitable culture media on the left:View Page
Koch's postulates include all of the following except:View Page
Which of the following media is a selective medium for Campylobacter jejuni:View Page
Which of the following organisms is most likely to be associated with gas gangrene:View Page
Which of the following is not true about members of the Enterobacteriaceae:View Page
Match the organisms on the left with their appropriate type on the right:View Page
Match organism on right to common name on the left.View Page
Sheep blood agar contains inhibitors to which of the following organisms:View Page
What is the purpose of using alcohol in the gram stain procedure:View Page
Which of the following best describes the organisms seen in this illustration:View Page
Which of the following best describes the organisms seen in this illustration:View Page
The Thayer-Martin agar plate seen in this illustration exhibits marked growth. The most likely organism found here would be:View Page
Which one of the following organisms is sensitive to bacitracin:View Page
The most likely organism to be cultured on the Lowenstein-Jensen agar slant illustrated here would be:View Page
The McFarland Comparison Card shown in the illustration is used to:View Page

Molecular Methods in Clinical Microbiology
Human Papilloma Virus (HPV) and Mycobacterium

Human papilloma virus (HPV) is estimated to be the most common sexually transmitted infection in the United States. Digene's hybrid capture assay for HPV received approval by the Food and Drug Administration (FDA) in 2003. Only in recent years have other manufacturers, such as Third Wave Technologies, added this virus to their testing capabilities.Mycobacterium species represented another desirable target for molecular testing. Although some improvements in cultivation and staining techniques had been realized through the incorporation of broth media and fluorochrome staining, identification is still hampered by the growth rate of the organism. Gen-Probe first marketed probes that would allow identification of tuberculosis, M. avium-intracellulare, and M. gordonae in culture positive specimens. These probes greatly streamlined the workup of culture positive specimens.Of great interest to both clinicians and infection control practitioners, is the direct detection of Mycobacterium in clinical specimens. Gen-Probe received FDA approval for its AMPLIFIED MTDâ product for this specific application (in smear positive specimens) in 1995. This method employs isothermal transcription mediated amplification; the amplicons are detected using the same hybridization as the culture confirmation tests.

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The Key Benefits: Improved Sensitivity of Detection

There are three key benefits that molecular methods can offer, in contrast to traditional culture methods:Improved sensitivity of detectionImproved specificity of identificationReduced turnaround timeImproved sensitivity of detectionSuccessful cultivation or detection of an organism depends on many factors, including the:Ability of the organism to survive transport Fastidious nature of the organism/its ability to grow in available culture media/systems Number of organisms present in a specimen Ability of a staining/culture system to visualize/recover low numbers of organisms Sensitivity of non-culture (antigen detection) methodsOrganisms that have been shown to be very labile are difficult to cultivate even when they are present in significant numbers. The immediacy of transport, plating, and incubation are critical factors that frequently cannot be controlled in a positive direction.Even under the ideal transport conditions, some organisms may require culture media and conditions that are not routinely available in every laboratory, which reduces the likelihood of successful cultivation.

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The Key Benefits: Specificity of Identification

Even if an organism can survive various transport conditions and will be recovered in available culture systems with reliability, identification protocols may not provide clear cut differentiation, based on phenotypic tests and characteristics. Sequencing techniques are increasingly utilized for certain classes of organisms. Good examples are the aerobic, non-spore forming gram positive rods, including Nocardia and related genera, and mycology isolates. A more routinely encountered application occurs in virology. Molecular methods allow for efficient typing of herpes simplex (type I versus type II) and subtyping of specimens positive for influenza A.

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Why can molecular methods offer improved turnaround times over cultivation methods? (Choose all that apply.)View Page
Challenges for Implementation: Required Work Skills

In addition to instilling a consciousness about movement from one area to the next, other work skills are critical and key to obtaining accurate results:Aseptic techniquePipetting skillsAseptic technique Due to the sensitivity of molecular assays, there is little margin for error. Cross contamination from specimens with large numbers of organism/target nucleic acid can result from the slightest deviation in aseptic technique. The use of aerosol barrier pipette tips and positive displacement pipette devices go a long way in preventing aerosol contamination, but do not replace the attentiveness of the technologist during each transfer of specimen material. When working with specimens, only one specimen container should be opened at a time.Pipetting skills Although more and more assays are being introduced with pre-prepared master mixes, invariably some reagent preparation is required. This often entails the transfer of very small volumes, which leaves no margin of error during pipetting. Attentiveness to accuracy of pipetting is a must.

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Polymerase Chain Reaction (PCR)

The polymerase chain reaction (PCR) copies DNA, utilizing repeated cycles of three basic steps. This reaction utilizes Taq DNA polymerase enzyme, which is a recombinant, thermostable DNA polymerase from the organism Thermus aquaticus. Step 1: DenaturationAfter an extraction process designed to release DNA from cellular material, an aliquot of the extracted sample is added to a reaction mixture which contains polymerase enzyme, forward and reverse primers for the target of interest, and nucleotides. During the first step, this mixture is heated (generally to 95°C). This causes complementary strands of DNA to separate (denaturation).Step 2: AnnealingThe reaction mixture is cooled to 55°C. During this annealing phase, if the target of interest is present in the patient sample, the primers will bind to their complementary sequences of DNA. Primers are short sequences of single stranded DNA that mark both ends of the target sequence. Two primers are utilized, one for each of the complementary single strands of DNA released during denaturation. The forward primer attaches to the start codon of the template DNA (the sense strand), while the reverse primer attaches to the stop codon of the complementary strand of DNA (the anti-sense strand). The 5' ends of both primers bind to the 3' end of each DNA strand.Step 3: Synthesis at 72°CThe temperature is raised, typically to a temperature of 72°C. At this temperature the polymerase enzyme begins the process of DNA synthesis. Free nucleotides, complementary to the bases in each strand, are added sequentially to both the sense and anti-sense strands. Synthesis always occurs from the 5' to the 3' direction on each primer. This results in the simultaneous synthesis of two new strands of DNA: in the direction from the start codon to the stop codon from the forward primer, and in the direction from the stop codon to the start codon from the reverse primer.These steps are repeated in cycles, resulting in a geometric doubling of the target sequence at the end of each cycle. A typical assay will utilize around 45 cycles. Once amplification is completed, detection and identification of the multiplied target occurs.

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Several methods of detection are available for the detection of Clostridium difficile in clinical samples. Which methods have the capability for detection in less than 48 hours? (Choose all that apply.)View Page

Multi-drug Resistant Organisms: MRSA, VRE, and Clostridium difficile
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.

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Staphylococcus aureus

Staphylococci are non-motile, non-spore-forming, gram-positive organisms occurring singly, in pairs, tetrads or in clusters resembling grapes. More than 20 species have been identified; three species are significant in their interactions with humans - S. aureus, S. epidermidis and S. saprophyticus.The staphylococci are members of the normal flora of the skin and mucous membranes of humans and warm-blooded animals. Colonization of the nares (nostrils) and skin can provide large reservoirs of organisms for transmission. Approximately 25-30% of the general population are colonized by Staphylococcus aureus, mainly in the nasal passages, but the organism can be found in most anatomical sites including the skin, oral cavity and GI tract.Infections are frequently acquired when the colonizing strain gains access to a normally sterile site as a result of trauma or abrasion to skin or mucosal surface. S. aureus infections range from superficial, localized skin infections, such as folliculitis, to deeper, more serious skin lesions and the more serious toxin mediated conditions – scalded skin syndrome and toxic shock syndrome.

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Staphylococcus aureus Virulence Factors

S. aureus is the most pathogenic member of the genus Staphylococcus; it possesses several factors that contribute to its virulence: Structural components of its cell wall function as a protective barrier, aid in adherence to mucous membranes, and allow the organism to resist phagocytosis. The production of several different toxins Enterotoxins A, D, F (TSST1) Exfoliative toxin ( causing scalded skin syndrome Cytolytic toxins (causing cell & tissue damage). Production of enzymes Catalase – distinguishes staphylococci from streptococci Coagulase – distinguishes S. aureus from other staphylococci Hyaluronidase & lipase – aid in skin colonization/infection spread Beta-lactamase – breaks down the beta-lactam antibiotics, e.g., penicillins, cephalosporins, carbapenems and monobactams.

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Clinical significance of Staphylococcus aureus

In general, the infection that develops is dependent on the virulence of the particular strain, the inoculum size, and immune status of the host. Staphylococcal infections are typically suppurative, producing abscesses filled with pus and damaged leukocytes surrounded by necrotic tissue. Skin infections range from superficial - boils, carbuncles and furuncles, to bullous impetigo; largely opportunistic infections that develop as a result of previous injury e.g., cuts, burns, surgical wounds - and scalded skin syndrome (extensive exfoliative dermatitis; also known as Ritter's Disease). Other major infections include pneumonia, osteomyelitis (localized infection of bone), and septic arthritis. S. aureus also causes food poisoning as a result of ingestion of food contaminated with an enterotoxin producing strain (enterotoxins A&D) and the potentially fatal toxic shock syndrome, a multisystem disease most often associated with the use of highly absorbent tampons. Toxic shock syndrome is attributed to another toxin (enterotoxin F – TSST1) released by certain strains of S. aureus.Human staphylococcal infections usually remain localized by the normal host defenses. Foreign objects (fomites) such as sutures or intravenous (IV) lines - are readily colonized by S. aureus from skin and can allow the organism to spread systemically via the blood stream – bacteremia/septicemia - leading to more serious infections. Staphylococcal pneumonia is becoming a frequent complication of influenza. Whatever the mode of entry, the invasive nature of S. aureus always poses the threat of more serious deeper tissue invasion and/or bacteremia and hematogenous spread.

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The pathogenicity of Staphylococcus aureus, as well as the frequency with which this organism produces infections, can be attributed to:View Page
Risk Factors

Medical conditions that lead to immunosuppression increase the risk of MRSA infection. Participating in contact sports, sharing towels or other personal items, living in areas with unsanitary conditions or living in crowded conditions, such as in dormitories or military barracks may also increase the risk of becoming infected with MRSA. Healthcare workers, the very young and the elderly are at increased risk of infection. Skin punctures and/or wounds increase infection risk by providing an entry point for the organism. Hospitalized patients are at risk of infection from healthcare workers with contaminated hands and from other MRSA carriers. Intravenous (IV) lines, surgical sites and implanted device can be easily contaminated with MRSA if infection control precautions are not followed.

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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.

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Laboratory Detection of Clostridium difficile

Several laboratory methods are currently available to aid in the detection of C. difficile including culture for toxigenic C. difficile (considered the "gold standard" for viable C. difficile detection), detection of Toxin A, B, or both, and molecular detection methods. These methods differ in their sensitivity and specificity and should always be used in conjunction with clinical considerations. To make the diagnosis, it is usually only necessary to submit 1-2 diarrheic (non-formed) stools per episode. Once positive for C. difficile by any laboratory method, there is no need for follow-up assays to make sure the organism or toxins are absent from the initial episode. If assays are performed for subsequent episodes, culture or tissue culture assay for Toxin B are probably most appropriate to avoid the possibility of detecting the initial antigen, toxin, or gene.

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Various methods have been employed for detection of C. difficile disease: cultivation of the organism, cell cytotoxin neutralization assays, and enzyme immunoassays have all been among the staples of diagnostic approaches. Which statements are accurate characterizations of these assays?View Page
Disinfection & Control of C. difficile Infection

C. difficile spores resist dessication for months and are known to persist on hard surfaces for up to 5 months. Spores persist even after exposure to air. Epidemic strain B1/NAP1/027 is known to hyper-sporulate, a virulence-associated characteristic of outbreak strains. Healthcare workers are an important vector for transmission as they may carry the spores on their hands or clothing. Alcohol-based hand sanitizers are very effective against non-sporulating organisms but do not kill C. difficile spores or remove the organism from the hands. The CDC recommends thorough hand washing using soap and water for care givers and family members alike.Patients with C. difficile infection (CDI) should be isolated to a single room with a bathroom or cohorted (roomed) together. Staff treating infected patients should use PPE (gowns & gloves) and wash hands after removing gloves. The use of gowns helps to prevent contamination of clothing. Surfaces should be decontaminated using a solution of 10% sodium hypochlorite (bleach), this is effective in reducing environmental contamination in hospital rooms. The CDC recommends the use of bleach for cleaning patient and staff rooms during outbreaks. Control strategies involving reinforcement of Infection control practices rather than drug restriction are more effective. These practices include: Proper education of staff members involved in care of CDI patients Better isolation compliance Use of gloves Frequent and thorough hand washing Environmental decontamination

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Clostridium Species

Clostridium are gram-positive or gram-variable, spore-forming, catalase-negative anaerobic bacilli. More than 100 species are currently recognized, though relatively few are encountered in properly collected clinical specimens from humans. There are three types of infection associated with Clostridium species: Non-invasive: Toxin-mediated Invasive: Progressive infection with tissue destruction Purulent disease: Closed space (e.g., in the peritoneal cavity) mixed infection with multiple organisms.Clostridium are well known as the agents of these classic toxin-mediated diseases : DISEASE TOXIN INVOLVED CAUSATIVE ORGANISM Tetanus or "lock jaw" Tetanospasmin Clostridium tetani Myonecrosis/Gas gangrene Exotoxins Clostridium perfringens Botulism (severe food poisoning) Botulin Clostridium botulinum

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Clostridium difficile

Most Clostridium infections arise from endogenous sources. That is, many of the Clostridium species that are associated with disease in humans are part of the normal intestinal microflora, which is true of Clostridium difficile.The organism was originally isolated in 1935 as a component of the normal intestinal flora of healthy newborns. It was dubbed difficile because the organism grows slowly and is difficult to culture. Early investigators also noted that the organism produced a potent toxin, but the relationship between C. difficile antibiotic-associated diarrhea (AAD) and pseudomembranous colitis (PMC) was not elucidated until the 1970's. PMC is an inflammatory disease of the colon caused by toxins of Clostridium difficile. Normal intestinal flora is an important factor in host response to an infectious microorganism. Resistance to intestinal infection is significantly reduced when there is a reduction in the normal flora as a result of antibiotic treatment. The most common manifestation of this decreased host resistance is the development of PMC.

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Pathogenisis of C. Difficile-Associated Diarrhea

Clostridium difficile is the leading cause of hospital-acquired diarrhea in the United States, with the number of cases rising annually over the last three decades. This is largely due to the increased frequency of antibiotic usage, the development of better detection methods, and the fact that hospital environments are increasingly contaminated with spores of C. difficile. The definition of C. difficile diarrhea includes > 6 episodes of non-formed diarrheic stool per 24 hours, along with prior antibiotic treatment. At least three events must occur in the pathogenesis of C. difficile-associated diarrhea (CDAD): Alteration of the normal fecal flora Colonic colonization with toxigenic C. difficile Growth of the organism with elaboration of its toxins"Colonization resistance" is the term used to describe the mechanism by which indigenous flora control overgrowth of C. difficile. This resistance may be compromised by the use of antimicrobial compounds, underlying illness, or therapeutic procedures. Infection begins with the ingestion of either the organism itself or spores, usually via the fecal-oral route. Spores in particular are able to survive the acidity of the stomach and germinate in the colon to produce vegetative organisms. Toxinogenic strains subsequently produce Toxin A, Toxin B, and/or the Binary Toxin leading to colitis, pseudomembrane formation, and watery diarrhea. Significant complications of the clinical disease associated with infection are hypoalbuminemia, toxic megacolon (acute toxic colitis with dilatation of colon), and pseudomembranous colitis (PMC).

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C. difficile disease is more likely to occur when:View Page

Mycology: Yeasts and Dimorphic Pathogens (retired 2/12/2013)
Arrange the fungal species that are listed in the drop-down box according to length of time of recovery in primary culture; from most rapid to the slowest.View Page
Match the complications that are most likely to be associated with each of the two yeast diseases that are listed in the drop-down box:View Page
The growth of the yeast-like colonies shown in the upper image was obtained on blood agar from a skin culture only in the area overlaid by virgin olive oil. The lower image is a photomicrograph of a lactophenol blue mount made from a portion of the colony. The disease associated with this fungus is:View Page
This photomicrograph is an acid-fast stained smear prepared from a yeast colony growing on ascospore agar. A helmet-shaped, red-staining, acid fast yeast cell is seen in the center of view at the tip of the arrow, against the background, blue-staining blastoconidia. The presumptive identification of Hansenula anomala was made. Predisposing conditions that may indicate that this isolate is more than a contaminant include:View Page
The ability of Candida albicans to alternate between two phenotypes, which may be related to the virulence of this species, is called:View Page
Each of the following is considered to be a virulence factor in Cryptococcus neoformans except:View Page

Normal Peripheral Blood Cells (retired 6/20/2012)
Cellular Immunity

Cellular immunity includes delayed hypersentivity reactions, graft rejection, graft-versus-host reactions, defense against intracellular organisms, and probably defense against neoplasms.Cellular immunity is mediated by lymphocytes which we call T-cells.T-cells are so named because they are dependent on the thymus for their production and development.The majority of T-cells are long-lived with an average lifespan of 4.4 years, but it is known that some survive for as long as 20 years or more.T-cells are capable of leaving and re-entering the circulation many times during their long life.T and B cells cannot be differentiated when viewing blood films.They are identified through the use of immunologic cell markers.

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OSHA Bloodborne Pathogens
You Are At Risk!

As a health care worker, you come into contact with materials that may contain bloodborne pathogens. These are infectious organisms, usually viruses, that live in human blood and body fluids.The bloodborne pathogens that are of greatest concern to health care workers are:Hepatitis B virus (HBV) Human immunodeficiency virus (HIV) Hepatitis C virus (HCV)

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OSHA Bloodborne Pathogens (retired)
You Are At Risk!

As a healthcare worker, you come into contact with bloodborne pathogens. These are infectious organisms, usually viruses, which live in human blood and other potentially infectious body fluids.The most important ones are... Hepatitis B Virus (HBV) Human Immunodeficiency Virus (HIV)

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Parasitology Question Bank - Review Mode (no CE)
Which of the following vectors has/have the capability of serving as a transport host for both the amebae and the flagellates?View Page
Match each parasite listed here with its corresponding infective stage:View Page
Arrange the following parasites in order according to life cycles from simple to most complex:View Page
The ameba that lives in the gumline of the mouth is known as:View Page
Which ameba listed here has a flagellate form as part of its life cycle?View Page
The trophozoite is the only morphologic form in the life cycle of:View Page
Which of the following parasites may be contracted by swimming in contaminated water?View Page
Houseflies are a possible transmission for which of these categories of parasites?View Page
Arrange the following life cycle phases of Diphyllobothrium latum in order beginning with human transmission:View Page
Arrange the following Plasmodium morphologic forms in order from immature to mature:View Page
Match each vector listed here with its respective parasite:View Page
Arrange the following phases of the Chilomastix life cycle beginning with human transmission:View Page
The adult worms of which of the following parasites reside in human intestine?View Page
Which of the following parasites have/has a life cycle that resembles that of the Plasmodium species?View Page
Match each parasite listed here with its respective mode of human transmission: (answers may be used more than once)View Page
The ELISA method may be used to detect:View Page
Serologic methods have been developed to identify which of these parasites?View Page
Match each organism listed here with the most likely specimen type where it may be recovered. Each answer may only be used once.View Page
Although not always the most practical, animal inoculation may be used to identify which of the following conditions?View Page
A specimen suspected of containing which organism may be cultured by placing it on an agar plate seeded with gram negative rods?View Page
This suspicious form measures 12 µm by 7 µm and was found in a stool sample.View Page
Recovered from a stool sample, this suspicious form measures 6 µm by 8 µm.View Page
This suspicious form measures 13 µm and was recovered from a stool sample.View Page
This suspicious form was recovered in muscle tissue.View Page
This suspicious form, shown here at both low (10X) and high dry (40X) power, measures 90 µm by 42 µm. It was seen in a stool sample.View Page
This parasite may inhabit the small intestine or take up residence in the bile ducts. It typically measures 145 µm by 75 µm.View Page
Recovered in a stool sample, this suspicious form measures 7 µm.View Page
This parasite was found on a blood smear.View Page
This suspicious form, found in urine, measures 120 µm by 50 µm.View Page
This suspicious form was recovered in stool.View Page
This suspicious form, found in stool, measure 10 µm.View Page
A 20 year-old female was admitted into the hospital complaining of 10 to 15 bloody mucous stools per day, fever, gastrointestinal disturbances, abdominal pain, and nausea. The preliminary O & P report went out as "Probable Entamoeba histolytica trophozoites and cysts, confirmation pending." This patient is most likely suffering from:View Page
Match each organism with its respective associated condition:View Page
Which parasite listed here is capable of crossing the placenta and causing serious harm to fetus?View Page
Immunocompromised patients, such as those with AIDS are at an increased risk of contracting which of the following conditions?View Page
A 32 year old male was seen in the emergency room with gastrointestinal discomfort. Upon questioning the patient it was learned that he first began feeling ill after spending a day at the park where he swam and played volleyball barefooted. He first noticed a lesion on his foot. Later, he developed vague respiratory symptoms. Now his largest complaint is severe abdominal pain along with occasional vomiting. This patient is most likely suffering from:View Page
This suspicious form, which was recovered in stool specimen and measures 32 µm, is responsible for which of the following diseases?View Page
This stool parasite measures 135 µm by 50 µm and is the causative agent of:View Page
This suspicious form, found in stool, which measures 15 µm by 10 µm, is responsible for which of the following diseases?View Page
This parasite, found in striated muscle, is responsible for which of the following conditions?View Page
This suspicious form is associated with which of the following conditions?View Page
The suspicious form pictured here is responsible for which of the following conditions?View Page
Perianal itching is the major symptom of infection with both forms of the organism pictured here. This parasite is the causative agent of:View Page
Which of the following parasites is the causative agent of swamp fever?View Page
This suspicious form, recovered in stool, measures 165 µm by 65 µm. It is responsible for causing:View Page
This suspicious form measures 15 µm and was recovered in stool. Which of the following conditions is/are associated with the presence of this form?View Page
Which of the following conditions may be associated with the presence of this stool parasite?View Page
With which of the following conditions is this suspicious form associated?View Page
Entamoeba, Endolimax, and Iodamoeba are members of which of subphylum?View Page
The class of protozoa with no apparent organelles for locomotion is known as:View Page
Which of the following parasites is/are considered as atrial ameba(e)?View Page
The motile, feeding stage of the amebas and flagellates is called:View Page
The protozoa are classified and placed in groups based on which of the following criteria? View Page
Trichomonas, Giardia, and Chilomastix belong to the subphylum:View Page
Binary fission is the means of reproduction for which of the following groups of parasites?View Page
Protozoal parasites that typically do not produce disease in humans are referred to as being:View Page
Match the following terms relating to the life cycle of select Sporozoa to their respective definitions:View Page
Match each parasite pictured with its respective classification:View Page
Match each parasite with its respective classification:NOTE: Answers may be used more than once.View Page
Match each amebic cyst listed here with its respective trophozoite form pictured:View Page
Match each parasite pictured with its respective classification:View Page
The rod-shaped structures that are believed to function as a food source for select amebic cysts and contain RNA are known as:View Page
The fever and chills syndrome associated with malaria is known as a/an:View Page
Which of these parasites are hermaphroditic in their adult phase?View Page
The body of a cestode is known as its:View Page
Match each parasite name listed here with its corresponding picture.View Page
Match each parasite listed here with its respective classification:View Page
Match each parasite listed here with its respective classification:View Page
The body of an adult cestode consists of segments called:View Page
Match each parasite listed here with its respective common name:View Page
Which of the following parasites lay live larvae?View Page
Dracunculus medinensis belongs to this category of parasites:View Page
Arrange the parasites listed here in order based on relative size from largest to smallest:View Page
Which of the following parasites is/are known to contain cytoplasmic ingested red blood cells in the trophozoite morphologic form?View Page
The nuclei of which of the following parasites lack peripheral chromatin?View Page
Arrange these parasites in order by general relative size from smallest to largest:View Page
Match each pair of parasites listed here with the key morphologic characteristics that help to distinguish between them:View Page
Thorn-like pseudopods are characteristic in trophozoite form of:View Page
The eggs of Necator americanus are basically indistinguishable from the eggs of:View Page
Match each parasite listed here with the key characteristic that aids in its identification:View Page
Arrange the following parasites in decreasing order (from large to small) based on relative size:View Page
Arrange the parasites listed here in increasing order (starting with none) based on the length of their undulating membranes:View Page
Match each parastie with the most common maximum number of nuclei present in the mature cyst form: (Answers may be used more than once.)View Page
The presence of two sporocysts each containing four banana-shaped sporozoites is characteristic of the oocysts of which of the following organisms?View Page
Match each picture with its respective morphologic form:View Page
Label the morphologic structures on this parasite form:View Page
Label the morphologic structures on this parasite form:View Page
Label the morphologic structures on this parasite form:View Page
Label the morphologic structures on this parasite form:View Page
Match each parasite morphologic form with its respective key morphologic key characteristic:View Page
Label the morphologic structures on this parasite form:View Page
A 55 year old female, who recently returned from an extensive trip to China, presented to her physician complaining of diarrhea and abdominal cramps. The doctor ordered a complete blood count (CBC), chem 21 panel, and stool for culture and parasite examination (O & P). The CBC revealed pronounced eosinophilia. The chem 21 and stool culture were unremarkable. The O & P revealed suspicious forms like the one below that each measured approximately 140 µm by 80 µm. This patient is most likely infected with:View Page
A 50 year old male domestic airline pilot was rushed to the hospital after complaining of tremendous fluid loss due to severe diarrhea. History revealed that the patient was diagnosed with AIDS 6 months ago. The doctor ordered a battery of tests including a stool for parasite examination. Since the sample was properly labeled indicating that the patient was immunocompromised, the lab performed both the standard processing procedures and a modified acid-fast (mod AFB) stain. The mod AFB stain revealed this suspicious form which measured a mere 4 µm. This patient is most likely infected with:View Page
A 12 year old female went to her doctor for her yearly back-to-school check-up. She was in good health and was asymptomatic at the time of the examination. Due to the increased incidence of parasites in the area, the doctor ordered a stool for parasite examination as part of the routine physical testing. Multiple suspicious forms, measuring approximately 9 µm each were seen. Which of the following is most likely the identification of these forms?View Page
A 58 year old male, who recently returned from an extensive overseas business trip to Africa, presented to the local clinic complaining of nausea, vomiting, and an achy feeling all over his body. At first he thought it was just the flu, but it persisted. The doctor ordered a battery of tests including blood smears for parasitic study. This suspicious form was recovered. The patient is most likely suffering from:View Page
An 18 year old immigrant from the Philippines presented to the local clinic shortly after relocating to the United States complaining of fever and chills. Examination of the young adult revealed enlarged lymph nodes. Blood was drawn and submitted for culture and parasitic examination. The culture was negative. This suspicious form was seen on the Giemsa-stained blood smear. It measures 225 µm in length. This patient is most likely infected with:View Page
A 65 year old Asian female presented to the emergency room exhibiting severe abdominal pain, fever and diarrhea. Examination revealed an enlarged liver that was tender to the touch. Patient history revealed that the woman worked in a fish processing plant for years prior to moving to the United States. Her diet was heavy in raw fish. Stool and duodenal contents were collected and sent to the laboratory for cultures and parasite examination. The cultures were unremarkable. This suspicious form was seen in both specimen types. It measures 27 µm by 14 µm. This patient is most likely suffering from:View Page
A 7 year old male presented to the local clinic experiencing mild, yet annoying, abdominal pain, diarrhea and nausea. Patient history revealed that the child lives in a shack where sanitation practices are marginal, at best. The dwelling is infested with rats and multiple insects. Stool was collected and submitted for parasitic study. Numerous suspicious forms, measuring 60 µm by 75 µm were seen. This child is most likely infected with:View Page
A 27 year old female graduate student recently returned from South America, where she completed a nature study of the rain forest. She spent months "living off the land." The woman went to her physician seeking treatment for a sinus infection, which she thought was responsible for several recent bouts of diarrhea. Upon questioning the patient, the doctor decided to collect stool for culture and parasitic examination. The stool culture was reported as "no enteric pathogens isolated." This suspicious form was seen on both wet preparations and on permanent stain. It measures 17 µm. The identify of this form is most likely:View Page
A fresh stool sample was submitted to the laboratory for parasitic examination on a 30 year old male who presented to a local clinic complaining of gastrointestinal discomfort and overall weakness. The only patient history available about the patient was that he was here job hunting and that he is originally from rural Mississippi. The sample was immediately processed and this suspicious form was seen. No other suspicious forms resembling eggs were seen. The patient is most likely infected with:View Page
A 10 year old male presented to the local Appalachian Mountain clinic complaining of vomiting, fever and severe abdominal pain. Patient history revealed that the child lives in the area in substandard conditions and receives only one balanced meal per day. A stool was collected and submitted for parasite study. This suspicious form, measuring 50 µm by 35 µm was found. This patient is most likely infected with:View Page
A 4 year old female from South Carolina was rushed to the emergency room who was suffering from malaise, bloody diarrhea and abdominal pain. Examination revealed rectal prolapse. Stool was submitted for parasitic examination and this suspicious form was seen. It measures 45 µm by 20 µm. Which of the following is the correct identity of this suspicious form?View Page
A 17 year old female went to her doctor complaining of diarrhea. With the exception of seasonal allergies, she was in relatively good health. Patient history was unremarkable. A stool was submitted for culture and parasite examination. The culture was reported out as "no enteric pathogens isolated." This suspicious form was seen on wet preparation and permanent stain. It measures 27 µm. This patient is most likely suffering from which of the following conditions:View Page
A stool collected at a local doctor's office was received in the laboratory for parasitic examination. The sample was not received in fixative and due to the new courier system did not arrive in the lab within the traditionally acceptable time frame. Due to logistical difficulties of the patient collecting and submitting another sample, the laboratory director authorized the sample to be processed. The comment "specimen delayed in transit, please evaluate results accordingly" was included in the report. These two suspicious forms were seen upon examination of the specimen. Label these two forms:View Page
A 6 year old female presented to the local clinic complaining of intense perianal itching and diarrhea. The doctor ordered a cellophane tape prep and stool for routine culture and parasitic examination. The cellophane tape prep revealed suspicious form on the left. The stool culture was negative. The form on the right was seen upon examination of the stool for parasites, which measures 10 µm. Label these two suspicious forms:View Page
I measure 12 µm and am found in stool.View Page
I am found in stool and may be easily mistaken for "junk." I measure 12 µm.View Page
I reside inside red blood cells, where I grow and grow until the cells are eventually destroyed.View Page
I measure 20 µm in length and am found in stool.View Page
The locomotive structures of Entamoeba histolytica are known as:View Page
This parasite, found in stool, measures 60 µm by 45 µm. Name that parasite!View Page
This suspicious form, that measures 25 µm, was recovered in an eye sample. It is associated with which of the following diseases?View Page
This parasite was recovered in a veterinary stool sample. Which of the following human parasites does it most closely resemble.View Page
This parasite measures 28 µm by 17 µm. Its common name is:View Page
Arrange the following phases of the Echinococcus granulosus life cycle in order beginning with human transmission:View Page
Which of the following parasites may be recovered in the peripheral blood?View Page
The episodes of fever and chills experienced by patients suffering from malaria are known as:View Page
The developmental morphologic stage in select arthropod life cycles in which the organism physically resembles the corresponding adult stage is called the:View Page
The presence of parasites in human blood is termed:View Page
This suspicious form, which measures 20 µm, was recovered in sigmoidoscopic material. Name that parasite!View Page
This suspicious form measures 18 µm and was seen in stool. Name that parasite!View Page

Phlebotomy
Discussion

This phlebotomist violated hospital procedures in several ways that could adversely impact patient care: Cleaning the site only with alcohol, not iodine, could result in a false-positive contaminated blood culture. This might result in the patient receiving unnecessary intravenous antibiotics, and could prolong the patients hospital stay unnecessarily. Drawing both cultures at the same time lessens the chance of recovering a bloodstream organism.Drawing both cultures from the same site might result in both of them being contaminated, making it very difficult for the physician to distinguish contamination from a "real" bloodstream infection.Relevant topics:Blood cultures: introduction, Avoid skin contamination, Blood culture site preparation 1, Blood culture site preparation 2

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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.

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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.

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Preliminary Identification of the Primary Select Agents of Bioterrorism
Bacillus anthracis

Clinical specimens where organism may be encountered: CSF Blood Stool (rare) Vesicle fluid, skin swab, or biopsy Gram stain morphology from clinical specimens: Large, gram-positive rods with square or concave ends in short chains Spores are usually NOT present Capsule may be viewed in smears from infected tissue, but this is NOT reliable Gram stain morphology from culture material: Large, gram-positive rods with square or concave ends, often in long chains (more than 2-4 cells) Cells easily decolorize as the culture ages Does NOT form capsules in culture Central to sub-terminal, oval spores, with NO significant swelling of the cell It must be noted that spore production increases with the age of the culture. Do NOT keep these cultures in the laboratory for longer than 24 hours for this reason!

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Yersinia pestis

Clinical specimens where organism may be encountered: Blood Lymph node aspirate Respiratory secretionsGram stain morphology: Gram-negative rod Resembles other Enterobacteriaceae Can form short chains Gram stains performed from blood culture or other liquid media may show bipolar staining (displayed by the arrows)Note: Use of Wright-Giemsa staining on direct specimen may enhance demonstration of characteristic bipolar staining, also referred to as "safety-pin" morphology. Use of this staining is of limited value, as the method is not very sensitive or specific.

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Francisella tularensis

Clinical specimens where organism may be encountered: Blood Biopsy, skin scraping, or swab Lymph node aspirate Respiratory secretions - oropharyngeal aspirate, sputum, or bronchial washingsGram stain morphology: Very tiny, gram-negative coccobacillus Pale or weak staining Due to the small size, often difficult to see individual cells

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Brucella species

Clinical specimens where organism may be encountered: Blood Bone marrow TissueGram stain morphology: Very small, gram-negative coccobacilli Stains very faintly and tends to retain crystal violet, especially in blood cultures May initially be identified as gram-positive Organism is larger than F.tularensis Individual cells are evident

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Gram stains are performed on positive blood culture bottles. Match the organism that MOST closely resembles the description of the Gram stain morphology provided.View Page
Yersinia pestis

Culture Characteristics: Growth at 22-25oC and at 35oC Organism prefers 25oC, so the colonies will grow faster at this temperature Growth occurs on most routine media, including sheep blood agar (SBA), chocolate agar (CHOC), MacConkey (MAC) agar, and routine blood culture media Non-lactose fermenter on MAC agar Growth is slower than other EnterobacteriaceaeColony Morphology on SBA at 35oC: At 24 hours, colonies are pinpoint and translucent with a gray-white color Colonies take on a yellow tint as they age, after 48-72 hours, referred to as a "hammered copper" appearance An irregular or "fried egg" appearance, shown in the lower image, can also be seen at 48-72 hours Characeristic Features: Growth at 22-25oC is a hallmark feature of this organism

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Which of the following organisms display the characteristic "Medusa head" on sheep blood agar (SBA) after 18 hours of incubation at 35°C?View Page
Yersinia pestis

Yersinia pestis is a dangerous, highly virulent organism that can cause laboratory-acquired infections. It should NOT be manipulated on an open bench.Catalase: Y. pestis is catalase positive. Catalase testing MUST be performed with extreme caution in a biosafety cabinet (BSC) due to the creation of aerosols. Oxidase: NegativeUrea: NegativeIndole: NegativeImportant note: Y. pestis is often incorrectly identified on automated identification systems. These systems often key out as Acinetobacter, Shigella, or an H2S negative Salmonella. If this organism is suspected, do NOT use an automated system for identification in order to prevent the creation of aerosols and misidentification.

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Francisella tularensis

Francisella tularensis is a dangerous, highly infectious organism that can cause laboratory-acquired infections. It should NOT be manipulated on an open bench.Catalase: F. tularensis is weakly catalase positive. Catalase testing MUST be performed with extreme caution in a biosafety cabinet (BSC) due to the creation of aerosols. Oxidase: NegativeBeta-lactamase: PositiveUrease: NegativeXV factors: Not required for growthImportant note: F. tularensis is often incorrectly identified on automated identification systems. These systems may key out as Haemophilus influenzae or Actinobacillus species.

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Brucella species

Brucella is a dangerous, highly virulent organism and the aerosols are highly infectious. It is the MOST common cause of laboratory-associated bacterial infections. Laboratory acquired cases have occurred by aerosol generating procedures, direct skin contact with cultures, and by sniffing cultures. It should NOT be manipulated on an open bench.Catalase: Brucella is catalase positive. Catalase testing MUST be performed with extreme caution in a biosafety cabinet (BSC) due to the creation of aerosols. Oxidase: PositiveBeta-lactamase: PositiveUrease: PositiveXV factors: Not required for growth (satellite phenomenon with S. aureus is negative)Serological testing: Often used because so difficult to grow. An acute and convalescent phase specimen should be collected 21 days apart.

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Burkholderia species

Burkholderia species is a dangerous and highly virulent organism that can cause laboratory-acquired infections. It should NOT be manipulated on an open bench.Catalase: Both organisms are catalase positive. Catalase testing MUST be performed with extreme caution in a biosafety cabinet (BSC) due to the creation of aerosols. Oxidase: B. mallei: Oxidase variable B. pseudomallei: Oxidase positiveIndole: Both organisms are indole negativeMotility: B. mallei: Non-motile B. pseudomallei: Motile

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Which of the following is NOT a characteristic of Burkholderia pseudomallei?View Page
When following sentinel laboratory procedures and protocols, any isolate that cannot be ruled out as one of the select agents should be immediately referred to your LRN reference laboratory.View Page
Location Where Organisms Naturally Occur, Disease Produced, and Mode of Transmission

These organisms can be encountered outside of a bioterrorism event and produce human disease. It's important to be familiar with the geographic areas where these organisms naturally occur and the how disease is transmitted.Bacillus anthracis: Bacillus species inhabit the soil, water, and airborne dust. Anthrax is the disease produced, which is transmitted to humans via direct contact with infected herbivorous animals. This is where the disease is primarily encountered. Anthrax is controlled in animals in the United States, so the disease is rare. In humans, most cases are cutaneous infections found in people that handle animals and animal products, including veterinarians and agricultural workers. Anthrax is consistently present in the animal population of some geographical regions, such as Iran and Pakistan, but only small numbers of animals experience the disease at any given time. Yersinia pestis: Y. pestis is found primarily in rodents, but can also be found in several animal species, such as cats, rabbits, camels, squirrels. Animal to human transmission most commonly occurs via a flea bite, causing the most common form of the disease known as the bubonic plague. Human-to-human transmission occurs by either flea bite or respiratory droplets. This causes an overwhelming disease known as pneumonic plague, which is the most likely form that would be implicated in the event of a bioterrorist attack. Human cases of the plague continue to occur in many countries, including Africa, the southwestern United States, parts of Asia, and the former Soviet Union. Francisella tularensis: Many animals, including rodents, rabbits, deer, and raccoons act as host for this organism. Humans and domesticated animals, such as horses, cattle, cats, and dogs can become infected. The infection is transmitted to domesticated animals by ticks and biting flies. Humans are most commonly infected from the bite of an infected tick or fly. Other means of infection include direct contact with the blood of infected animals when skinning game, eating contaminated meat, drinking contaminated water, or inhaling the organisms produced by aerosols. F. tularensis carries a high risk of laboratory acquired infection and documented cases of infection have occurred. Most cases of tularemia are reported in the southern and south-central United States.

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Location Where Organisms Naturally Occur, Disease Produced, and Mode of Transmission, continued:

Brucella species: Brucella is distributed in nature worldwide and found in domesticated and wild animals, such as cattle, sheep, and pigs. Infection with Brucella species, known as brucellosis, is caused in humans by exposure to infected animal fluids or food products. This includes ingesting non-pasteurized dairy products, such as milk or cheese, inhaling aerosols, and skin contact with the fluids of infected animals. Brucellosis poses an increased risk of occupational exposure to laboratory, veterinary, and slaughterhouse workers. Brucella is the most commonly reported laboratory-associated bacterial infection.Burkholderia mallei and B. pseudomallei: Most Burkholderia are found in soil, but B. mallei is only found in mammals. B.mallei is the causative agent for Glanders which primarily affects animals such as donkeys, mules, and horses. Horses, the organism's natural host, are highly susceptible to infection. Human infection is rare and usually occurs in people working with infected animals or laboratory workers handling the organism. The organism is endemic in Africa, Asia, the Middle East, and Central and South America, and usually enters via the eyes, nose, mouth, abrasions or cuts in the skin, or through inhalation. B. pseudomallei is found in soil and water and can accidentally infect animals, plants, and rarely humans. It is the causative agent of melioidosis, which is endemic in areas of southeast Asia, Taiwan, and northern Australia. The organism generally enters through cuts in the skin, ingestion of contaminated water, or by inhalation of an aerosol.

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Match the organism to the disease produced outside a bioterrorism event.View Page

Reading and Reporting Gram Stained Direct Smears
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."

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This Gram stain was prepared from a sputum specimen and is viewed under oil immersion (1000X). What is the structure that is indicated by the arrow?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.

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Reading Gram Stained Direct Smears
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

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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.

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Contaminated Gram Stain Solution

Contamination of the staining solutions rarely occurs, but should be suspected when smears repeatedly contain the same organisms, and these organisms do not grow or are inconsistent with the clinical picture. Yeast and gram negative rods can occur as stain contaminants.

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Special Considerations for Genital Smears

Gram negative cocci which occur in pairs with their adjacent sides flattened, giving them a coffee bean appearance, are typical of the genus Neisseria. Neisseria gonorrhea is commonly found within white blood cells; these organisms must be carefully sought within genital tract specimens, and their presence or absence should be specifically noted in the report.The presence of intracellular gram negative diplococci in a purulent male urethral smear is presumptive evidence of gonococci, but this assumption cannot be made in a female.Beware of other organisms which resemble gonococci but are found extracellularly, such as Acinetobacter lwoffi, which must be distinguished biochemically.Direct smears read specifically for the presence of gonococci should include a direct reference to gram negative intracellular diplococci.

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Reading Gram Stained Smears From Cultures (retired 2/12/2013)
Which of the following statements are TRUE for the Gram positive control smear?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.

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Variations in the Ends of Rods

The ends of rod-shaped organisms may be rounded or tapered.

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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.

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Information to be reported

The following information is reported: Gram stain reaction ShapeThe cellular arrangement is not usually included in the report because it may vary, depending on the culture medium (liquid or solid) from which the organism was taken.

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Culture Smears: Importance and Reporting

The culture smear is used to determine the staining characteristic, size, shape and cellular arrangement of the unknown organism. This data helps the microbiologist to decide on additional culture and identification methods. By correlating the Gram stain reaction, size, shape, and cellular arrangement of the organism with colony morphology and growth requirements, the microbiologist may be able to tentatively identify the organism. This information may help the physician to optimize treatment until definitive culture and antibiotic susceptibility results become available. Gram stain reaction and bacterial shape must be included in the report.The cellular arrangement is usually not included in the report since it may vary depending on the culture medium (liquid or solid) used to isolate the organism. The following 12 screens contain additional ungraded practice questions pertinent to the material covered.

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Observations that can be of value to the microbiologist include:View Page
The process of microorganism identification includes: (Choose ALL of the correct answers)View Page

Real-Time PCR
DNA Polymerase

Polymerases are enzymes that synthesize DNA from an existing template. Polymerase requires a primer, nucleotides, and magnesium in order to function. In early PCR methods, the DNA polymerase was inactivated during the denaturation step. This required new polymerase to be added during each cycle of PCR. This problem was solved by the discovery of an enzyme termed Taq polymerase. Taq polymerase is isolated from the thermophilic organism Thermus aquaticus, a natural bacterium found in thermal springs. The Taq polymerase has optimal activity at 72°C but it can survive in temperatures up to 95°C. Today there are several other kinds of thermostable enzymes that are available for PCR, such as Pfu and Tli DNA polymerase. However, Taq is the DNA polymerase that is used most often in PCR procedures.

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Microbe Detection

Because real-time PCR can amplify extremely small amounts of genetic material, it can be used to detect and identify minute numbers of microbes in a specimen or in the environment. It can even be used to detect DNA of microorganisms that are nonviable or difficult to grow in culture. Before PCR and other amplification methods, microbes had to be cultured in order to obtain enough genetic material to analyze. Thus, dead microorganisms were almost impossible to identify. Cloning for microbial DNA was the method generally used to garner genetic material from microbes that were hard to grow; this process can be extremely lengthy and time consuming. The use of PCR creates a fast and practical way to replicate microbial genetic material, whether alive or dead. PCR is also commonly used for microbial fingerprinting in outbreak investigations as a way to confirm transmission or find the outbreak source. One can look for repeats in the genetic sequences by amplifying random polymorphic DNA, yielding PCR products of various sizes. These products can then be run through an electrophoresis gel, creating a band pattern that is known as the DNA fingerprint. PCR can also be used to fingerprint an entire microbial community in lieu of a specific organism. A community fingerprint is created by amplifying a sequence that is found in all organisms of interest. Community fingerprints can be compared over time to identify stability and variations. PCR has an advantage over culture techniques due to the differences in growth requirements and growth time. With cultures, it is common to miss organisms and determine relative abundance and frequency.

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The Urine Microscopic: Microscopic Analysis of Urine Sediment
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 high power field (HPF) are seen, the bacteria are considered to be clinically significant.

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Trichomonas under Phase-Contrast

Under phase contrast, Trichomonas organisms appear much darker than the surrounding white blood cells (WBCs).

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Transfusion Reactions
Bacteria Implicated in Contamination

Yersina entercolitica is most likely responsible for septic reactions in transfusions of Red Blood Cells. This organism is usually acquired by ingestion of contaminated food and causes mild symptoms of abdominal pain and diarrhea. Growth of Y. entercolitica is enhanced in iron-rich environments such as red blood cells. Other organisms reportedly found in Red Blood Cell units are Campylobacter species, Serratia species, Pseudomonas species, Enterobacter species, and Echerichia coli. These bacteria can produce endotoxins which cause a reaction in the patient. The majority of organisms associated with platelets transfusions are normal skin flora. Staphylococcus aureus, coagulase-negative staphylocci, aerobic and anerobic diptheroid bacilli, streptococci, and gram-positive bacilli are frequently isolated. Some transfused organisms have been implicated in a delayed post-transfusion illness. Pseudomonas aeruginosa and Burkholderia capacia have been isolated in cryoprecipitate and plasma. These organisms grow optimally at 30oC and have been found in water baths, accentuating the importance of overwrapping components that are thawed in a water bath. Rickettsia organisms are intracellular bacteria which are transmitted by ticks or insects. These bacteria are the causes of Rocky Mountain spotted fever, ehrlichiosis, and scrub typhus, and may be transmitted by transfusion. Similarly, the organism which causes Lyme disease may be transmitted as well. There have no reports of either of these organisms transmitted by transfusion.

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Tuberculosis Awareness for Health Care Workers
Tuberculosis Infection

TB infection is usually followed by an immune response and latency after exposure. In about 5-10% of cases, the latent period progresses to an active infection.Infection occurs when a susceptible person inhales droplet nuclei containing Mycobacterium tuberculosis and the organism reaches the alveoli of the lungs. The minimal infectious inoculum may be as low as one viable organism.About 2-12 weeks after infection, the immune system limits multiplication of additional bacteria and the immunological test becomes positive.Latent tuberculosis infection (LTBI) is the stage when the viable organism remains in the body; the individual has no symptoms and is noninfectious.Most persons infected with M. tuberculosis do not experience clinical illness and are noninfectious. About 5-10% of persons who are infected and are not treated will develop active TB during their lifetime. The risk for progression is highest during the first several years after infection.Most often, M. tuberculosis infects the lungs. However, it can infect almost any organ in the body, including bones and joints. Tuberculosis meningitis is a TB infection that occurs outside the lungs with devastating consequences, most often in young children and patients with AIDS.

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How Tuberculosis is Spread

Mycobacterium tuberculosis is spread through infectious droplet nuclei. When a person infected with pulmonary tuberculosis coughs, sneezes, shouts, or sings, the infectious particles are expelled into the air. The risk of infection is related to both concentration of infectious droplet nuclei and duration of exposure. Laboratory workers are at risk when an infectious aerosol is generated while handling liquid cultures, during preparation of frozen sections, and when performing autopsies on infected patients.

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