What are some symptoms that can tip you off as a doc that an infection may be present in a patient?
rash fever headache weakness cough seizure diarrhea vomiting myalgia blurred vision pain nausea skin ulcer arthritis inflammation
What is the process of laboratory diagnosis of bacterial diseases?
- physician suspects infectious disease.
- physician starts appropriate treatment
- physician collects specimen & puts it in proper medium.
- physician gives preliminary clinical diagnosis to microbiologist-they need to know what they’re looking for.
- Microbiologist selects diagnostic method & reports results.
- Physician adjusts treatment.
T/F A physician should never begin therapy (including antibiotic therapy) w/o having confirmation from the microbiologist of the causative organism.
False! usu you do begin treatment b/c you might not get results back for days or even a week! They need treatment before then!
T/F A physician should never tell the microbiologist of their suspected diagnosis for fear of biasing the scientist.
FALSE. There are so many organisms-it is helpful to know what you’re looking for. You work as a team!
What are possible infectious organisms?
bacteria fungi parasites viruses **don't always know which one you are dealing with.
What is the most common reason for failing to give an etiologic diagnosis w/ infectious disease?
improper specimen collection
for bacterial infections: distinguishing normal flora from those causing an infection.
What is a direct specimen?
microbes are in sterile site that can be accessed directly (e.g., needle aspiration of deep abscess, or blood collection)
blood culture if you suspect bacteremia
What is an indirect specimen?
microbes are in sterile site but must be collected through a non-sterile site (e.g., voided urine sample)
What is a contaminated specimen?
Microbes are in site contaminated with normal flora (e.g., throat or stool culture)
What is the most common tool used for specimen collection? What are the problems with this?
problems: only collects a small amount & easily dries out.
What is the importance/purpose of the transport media for specimen collection?
Transport media (i.e., buffered fluid or a semisolid medium) prevent drying out, maintain neutral pH, and minimize growth of contaminants
When do you want the transport container to be aerobic or anaerobic?
aerobic if the organism is aerobic.
anaerobic if the organism is anaerobic
**you want the organism to still be alive so that it can be cultured & identified.
A 34-year-old man presents with a red, swollen, and painful left knee. There is evidence of a small cutaneous wound, and the patient indicates he punctured the skin while taking down an old shed two days before. The lesion has evidence of crepitus (gas in the tissue). Fluid is aspirated from the lesion, placed it in an anaerobic transport vial, and sent to the lab. Select the anaerobic pathogen that is under suspicion.
a. Staphylococcus aureus
b. Clostridium perfringens
c. Pasteurella multocida
d. Eikenella corrodens
e. Streptococcus pyogenes
B. Clostridium Perfringens **this is the only anaerobic organism listed here it causes gas formation common skin lesion infection if left untreated--gangrene.
What are some ways that you can identify specific microbes in clinical microbe lab?
Microscopy Broth and Agar Culture (antibiotic sensitivity testing) Biochemical Characterization Antibody Detection Antigen Detection Nucleic Acid-Based Tests
What are 3 ways to directly examine a specimen?
What is bright field microscopy?
light focused directly on specimen (most common)
What is dark field microscopy?
central light is blocked, peripheral light only collected as scatter from microbes
What is fluorescence microscopy?
similar to darkfield, except microbes are labeled with dye that fluoresces when it interacts with light of an appropriate wavelength
When do you really want to use dark field microscopy?
when specimens are thin & difficult to see
Most bacteria are viewed how?
via brightfield microscopy
100X magnification w/ oil immersion
What are the different stains used for identifying bacteria?
What are simple stains? Give some examples.
use a single dye to visualize bacteria
Ex: crystal violet or methylene blue
for E coli or cornyebacterium
What are differential stains? Give some examples.
are used to distinguish different bacterial groups (e.g., gram-positive vs. gram negative)
also acid fast.
What are special stains? Give some examples.
are used to detect bacterial structures (e.g., capsules, flagella, and endospores)
Ex: india ink to see large sugar capsule on fungus that produces fungal meningitis
Which steps do gram staining & acid fast staining share?
After an acid fast stain…what color are acid fast organisms? Which condition requires an acid fast stain?
Acid fast appear red.
What’s the deal with bacterial cultures?
Culture of microbes in nutrient broth or on agar medium is commonly used
A single microbe can grow to amounts that are visible (e.g., turbidity in broth or colonies on agar)
Antibiotic susceptibility testing can be done on agar plates
T/F Almost all medically important microbes can be cultured.
What are some different potential shapes of bacterial colonies on agar?
circular irregular filamentous rhizoid raised convex flat umbonate crateriform undulated margin filiform margin curled margin lobate margin
Describe a nutrient medium.
– made from animal or plant products supplemented with a variety of nutrients (grow many microbes, but there is no “universal” culture medium)
Describe a selective medium.
used when specific pathogens are sought in sites with an extensive microbial flora. Usually chemical additives or antimicrobials that inhibit unwanted microbial growth
Describe an indicator medium.
contain substances designed to demonstrate biochemical or other features of specific pathogens (i.e., pH indicators of fermentation of specific sugars, or red blood cells that can be hemolyzed)
If you have a contaminated specimen from a stool sample b/c you are focused on a diarrheal disease…which medium should you use?
so that you can halt the growth of random flora.
What is an example of something that can be grown on a nutrient medium?
What are some examples of selective/indicator media & the things grown on them?
MacConkey Agar: E coli v. Proteus mirabilis
Eosin Methylene Blue Agar: E coli
Hektoen Enteric Agar: Salmonella v. shigella
What are some of the factors that you need to consider with culture conditions?
broth v. solid media
aerobic v. anaerobic
What are capnophilic organisms?
grown in air, but still require CO2 for growth
Describe the culture of most aerobic bacteria.
Cultures of most aerobic bacteria are maintained in an incubator at 35 to 37°C. Some bacteria grown in air but require CO2
What is the purpose of antimicrobial sensitivity testing?
Allows selection of most effective chemotherapeutic agent against the bacterial isolate
What are 2 tests that are often used to find the MIC of an antibiotic?
Broth dilution tests and agar diffusion tests are routinely used to determine the minimum inhibitory concentration (MIC) of an antibiotic
T/F The broth dilution method has been fully automated in the clinical microbiology lab.
What are some common biochemical reactions used to identify bacteria?
Bacitracin A disk Bile solubility Catalase Coagulase Hippurate Hydrolysis Indole A rapid test Optochin P disk Oxidase PYR hydrolysis
What’s the deal with the bacitracin A disk?
identifies strep pyogenes
sensitive to low conc’n of bacitracin
What’s the deal with the bile solubility test?
differentiates pneumococci from streptococci
pneumococci are bile soluble
What’s the deal with catalase tests?
defines major groups of bacteria
Catalase Positive: staphylococci
Catalase Negative: streptococci & enterococci
What’s the deal with the coagulase tests?
staph aureus is coagulase positive
What’s the deal with the hippurate hydrolysis test?
positive-group B strep
What’s the deal with the indole A rapid test?
E coli ID from urine
What’s the deal with the optochin P disk?
if susceptible–points to strep pneumonia
What’s the deal with the oxidase test?
amongst gram negative rods–some are oxidase + & some are oxidase -
What’s the deal with PYR hydrolysis?
helps ID S. progenies & enterococcus
T/F One way to perform biochemical characterization of pathogens is the API20e system.
Antibodies formed in response to most bacterial infections can be detected by methods such as…name 3.
ELISA (Enzyme Linked Immunosorbent Assay)
How could the antibodies present help you to determine the timing of an infection?
IgM: more recent infection
IgG: old or chronic infection
Antibody Detection is also known as? What is it most useful for?
Useful For: Epidemiologic Studies
When would you not want to use antibody detection for diagnosis?
in immunocompromised patients!
Like a SCID kid. They can’t make antibodies.
What are some tests that can be used for antigen detection? What is an example of a rapid antigen test?
Antigen Detect: immunofluorescence test
Rapid Antigen Test: Rapid Strep Test
What are the guidelines for reliability of a rapid strep test?
If it is positive-reliable. Treat it.
If it is negative–you can’t be sure. You should culture it. If positive, treat it.
What is pharyngitis usually caused by?
viruses, not bacteria
A 15-year-old female presents with fever and sore throat. Upon examination, the pharynx is bright red and swollen. A rapid Strep antigen test is performed and the results are negative. This is not unexpected as most pharyngitis is caused by viruses. However, given the high false negative rate of rapid antigen tests, how do you proceed?
a. Send the patient home with a prescription for penicillin
b. Get a throat culture and wait for the results before prescribing penicillin
c. Retest the patient with the rapid antigen test
d. Get a throat culture and prescribe penicillin just in case
e. Immediately perform a tonsillectomy
B. Throat Culture & then treat. B/c of all of our antibiotic resistance.
What are the 2 categories of nucleic acid analysis?
Describe how non-amplified tests work for nucleic acid analysis.
based on hybridization of nucleic acids from pathogens to labeled probes. These rapid assays are usually used to identify cultured bacteria where sensitivity is not as important
Describe how amplified tests work for nucleic acid analysis.
(PCR)-based tests are the amplified assays, and are the most commonly used in the diagnostic laboratory. These tests are extremely sensitive but subject to contamination; great care is needed to run good PCR tests
Once again, which is more sensitive…amplified or non-amplified nucleic acid analysis?
What are the basics of how PCR works?
You heat to separate strands. You hybridize. Heat Hybridize. Thus, you get these amplified results from one specimen.