Isolation and Identification of Bacterial Pathogens Flashcards
What is the role of the clinical microbiologist in medicine?
To isolate and identify the causative agent of an infectious disease within 48 hours of receiving a specimen obtained from the patient.
Why is early identification of the causative agent of an infectious disease important?
Because it can:
- minimize the severity and duration of a disease if proper treatment can be initiated immediately
- identify the most suitable antibiotic for treatment of the infection
- identify the causative agent of a disease outbreak in a hospital setting (i.e., contaminated supplies, poor aseptic technique by hospital staff).
Describe the use of microscopy in the isolation and identification of bacterial pathogens.
Preliminary diagnosis of only a few bacterial pathogens can be identified from clinical specimens using specific staining techniques and microscopy.
Sometimes this technique is a very important diagnostic tool because it may not be possible to cultivate the bacterium outside of the host.
However, the specimen collected during the clinical examination of a patient may contain several microorganisms, and microscopic examination may not be able to identify the agent of disease.
In these cases, the specimens are used to inoculate growth media for further tests.
What is the primary isolation media generally used for growth-dependent identification?
The primary isolation media is often a general purpose media such as blood agar.
Blood agar supports the growth of most aerobic and facultative anaerobic bacteria.
Enriched media may be required for the culture of metabolically fastidious bacteria.
Enriched media contains specific growth factors which are often required by certain pathogens.
During growth-dependent identification, what is an important first step?
After incubation for one to several days, isolated colonies appear on the surface of the agar plate.
Each isolated colony consists of millions of bacterial cells.
Observation of these colonies for size, texture, color, and hemolytic reactions (if grown on blood agar) are an important first step in bacterial identification.
Whether or not the organism required oxygen for growth is another important differentiating characteristic.
Identification of a clinical isolate can frequently be made using a variety of growth-dependent assays.
What are these methods based on?
Morphological and metabolic characteristics of bacteria.
Differentiate between selective media and differential media.
Selective media is one in which compounds are used to inhibit the growth of certain bacteria, but not other types of bacteria.
Differential media is one in which an indicator dye allows identification of bacteria by their appearance on the growth media.
Some growth media are both selective and differential.
What is a simple method to determine the antibiotic sensitivity of a clinical isolate?
The disk diffusion antibiotic susceptibility test
A zone of the inhibition of bacterial growth is observed if the bacteria are sensitive to an antibiotic; if the bacteria are resistant to the antibiotic, they continue to grow near the paper disk.
In the sample image, the bacteria are sensitive to all antibiotics except #4 and #6.
What is the size of the zone of inhibition dependent on?
How does this zone determine whether an organism is sensitive or resistant to an antibiotic?
The diffusion rate of the antibiotic, the degree of sensitivity of the microorganism, and the growth rate of the bacterium.
The test is performed under standardized conditions and standard zones of inhibition have been established for each antibiotic.
If the zone of inhibition is equal to or greater than the standard, the organism is considered to be sensitive to the antibiotic.
If the zone of inhibition is less than the standard, the organism is considered to be resistant.
Certain human pathogens cannot be isolated in the laboratory, or grow extremely poorly in culture.
Successful isolation can be slow and in some instances impossible.
Describe growth independent identification methods.
Many of these detection methods involve the use of antibodies that recognize determinants expressed on the surface of the pathogen.
In some protocols, the assays are used to directly detect the presence of the pathogen; in other protocols, the assays are used to detect the presence of antibodies to the pathogen from the patient’s serum.
For example, agglutination assays to detect group A streptococcus infections can be done in the doctors office. The patient’s throat is swabbed and streptococcal antigen is extracted directly from the swab (without prior bacteriological culture). The diagnosis can be confirmed by observing the agglutination of latex beads coated with antibodies that recognize the bacterial antigen of group A Streptococcus.
Another example involves the use of fluorescently-labelled antibodies. These antibodies would recognize surface antigens on the pathogen. They can be added directly to the sample (e.g., tissue from a lung biopsy), and if the pathogen is present, a positive identification can be made by visualizing the fluorescently-labelled bacteria under the microscope.
Describe how dependent and independent identification methods may be used in practice.
In many cases, the identification of a bacterial pathogen involves the initial isolation and identification using growth-dependent methods, with confirmation using serological assays (growth-independent methods).
