ASEPTIC TECHNIQUE
ASEPTIC TECHNIQUE: In handling bacteria specimens, do everything possible to assure that extraneous bacteria from you do not contaminate the specimen. This is not sterile technique.
Preparation and Staining:
Heat Fixing: After air drying a slide, applying gentle heat helps the bacteria to adhere to the slide.
Simple Stain (Methylene Blue): Stains all cells and can illuminate morphology.
GRAM STAIN:
PRIMARY STAIN: Crystal Violet stains both Gram+ and Gram- and makes all bacterial cells appear purple.
Both Gram+ and Gram- will stain with this dye.
CHELATING AGENT: Add Gram's Iodine which will adhere only to cell walls that have a lot of peptidoglycan in them (i.e. Gram+)
DECOLORIZATION: Add Acetone very briefly to wash away the Crystal Violet dye.
Gram-: The purple dye will be washed away.
Gram+: The purple dye will remain adherent.
COUNTERSTAIN: Then add Safranin which will stain cells pink.
GRAM-POSITIVE: Will appear purple (from Crystal Violet) -- Safranin doesn't stain it.
GRAM-NEGATIVE: Will appear pink, as it takes up the Safranin.
FALSE GRAM-NEGATIVES: Dead Gram+ cells will appear as though they are Gram-. In antibiotic-treated specimens, a certain portion of such cells is expected.
ACID-FAST STAIN: Stains Mycobacteria such as Mycobacterium Tuberculosis.
LIPID cell wall gives them the property of weak initial staining, plus strong retention of initial dye (like Gram+) once stained.
PROCESS:
Carbol-Fuscin dye is used initially, with heat, for 20 to 30 minutes (long time)
Acid-Alcohol is then used as a decolorizing agent.
If the organism resist the decolorization, it is considered acid-fast.
FLUORESCENT STAIN:
It has the advantage that you can scan at low power until you find something that fluoresces, then you can hone in on it.
IMMUNODIAGNOSTICS
Hemagglutination Inhibition: Certain organisms can agglutinate red blood cells. If you have antibodies to impede that agglutination, then you have a positive test result (antibody is present).
Direct Fluorescent Antibody: Fluoro tagged antibody checks for presence of antigen directly.
Indirect Fluorescent Antibody: First allow antigen-antibody reaction, then use a second Fluoro tagged antibody (anti-antibody) to identify presence of the first antibody.
Complement Fixation: Usually avoided; expensive.
ACUTE and CONVALESCENT TITERS: Take an antibody titer at initial infection (acute), and again four to five weeks later. You would expect a four-fold increase in titer if the suspected organism is indeed the infecting organism.
There is usually a baseline level of antibody present with or without infection, because of cross-reactivity with other antigens. Thus you use acute:convalescent titers as a means of comparison.
For some organisms, there are also absolute antibody levels that are diagnostic.
Radio-Immunoassay (RISA, ELISA): Take the patient's direct specimen and try to detect residual evidence of the organism's presence. Used when recovery of bacteria itself is not possible.
Molecular Probes
Polymerase Chain-Reaction (PCR): Can also be used for direct detection but it's expensive.
CULTURE:
EXCEPTIONS to the fact that organisms can be cultured in 24 hours.
Mycobacterium Tuberculosis takes up to eight weeks to culture.
Syphilis and Leprosy organisms have never been cultured.
Streaking for Isolation: Process of inoculating a plate such that you get single bacterial colonies once you culture the plate.
PROCESS: Take an inoculating stick, flame it, dip it into the bacteria (tissue sample), and then streak across the plate. Then flame it again, and streak it again.
SUSCEPTIBILITY TESTS:
KIRBY-BAUER METHOD of Antibiotic Susceptibility Testing: Testing is done using the Disk-Diffusion Method
PROCEDURE:
Grow confluent bacterial colony on plate
Add antibiotic disc in center.
Measure the Zone of Inhibition (diameter) to determine bacterial susceptibility.
RESULTS: The larger the zone of inhibition, the more susceptible are the bacteria.
Resistant: No zone of inhibition is found.
Intermediate: Intermediate zone of inhibition.
Susceptible: Larger zone of inhibition.
An interpretive table or computer, along with known concentrations of antibiotic, must be used to interpret results.
E-TEST: ANAEROBE Susceptibility Test. Strict Anaerobes must be cultures under special conditions.
Instead of a disk, the antibiotics are contained in capillary disks.
SPIRAL GRADIENT ENDPOINT TEST: Another susceptibility test
Line several isolates (can be from different patients) up on a single plate in wheel-spoke fashion.
Growth inhibition at each line can then be observed and interpreted by computer.
SERUM BACTERICIDAL TEST: Susceptibility test for patients that are extremely sick. Take serum sample and test amount of antibody actually in patient's serum. Then plate that antibody out onto culture and verify that it actually does kill the bugs at its peak and trough concentrations in the patient's serum.
A 1:8 dilution of serum inhibiting growth should be indicative that antibiotics are effective enough to kill bacteria in vivo.
MINIMUM INHIBITORY CONCENTRATION (MIC): The minimum amount of antibiotic necessary to inhibit bacterial growth to the point that no bacteria are visible with the naked eye.
This doesn't necessarily mean that no bacteria are there at all. Healthy person should be able to handle this bacteria-level with normal host-defenses.
PROCEDURE:
Take test-tubes containing varying concentrations of antibiotic, plus a control tube with no antibiotic.
Add growth media and 107 bacteria to each tube.
The most dilute tube in which no turbidity is seen is the MIC -- the minimum antibiotic concentration that inhibits bacterial growth.
MINIMUM BACTERICIDAL CONCENTRATION (MBC): Amount of antibiotic necessary to kill 99.9% of all organisms. This value must be used when treating immunocompromised patients.
PROCEDURE: To determine MBC, we must take all of our clear tubes from the MIC and culture them onto plates. Then, the plate on which the fewest number of colonies grows is termed the MBC.
Antibiotic Tolerance occurs when an isolate has a serial difference of five tubes between its MIC and MBC. Normally, the difference is only one or two tubes. This translates to a 32-fold difference in concentration.
CHOCOLATE AGAR: Heat-lysed red-blood cells in agar.
HEMOLYSIS: Appearance of culture in blood agar.
alpha-HEMOLYSIS: Partial hemolysis, appearing green.
beta-HEMOLYSIS: Full hemolysis. A "halo" appears around the colony.
gamma-HEMOLYSIS: No hemolysis.
Preparation and Staining:
Heat Fixing: After air drying a slide, applying gentle heat helps the bacteria to adhere to the slide.
Simple Stain (Methylene Blue): Stains all cells and can illuminate morphology.
GRAM STAIN:
PRIMARY STAIN: Crystal Violet stains both Gram+ and Gram- and makes all bacterial cells appear purple.
Both Gram+ and Gram- will stain with this dye.
CHELATING AGENT: Add Gram's Iodine which will adhere only to cell walls that have a lot of peptidoglycan in them (i.e. Gram+)
DECOLORIZATION: Add Acetone very briefly to wash away the Crystal Violet dye.
Gram-: The purple dye will be washed away.
Gram+: The purple dye will remain adherent.
COUNTERSTAIN: Then add Safranin which will stain cells pink.
GRAM-POSITIVE: Will appear purple (from Crystal Violet) -- Safranin doesn't stain it.
GRAM-NEGATIVE: Will appear pink, as it takes up the Safranin.
FALSE GRAM-NEGATIVES: Dead Gram+ cells will appear as though they are Gram-. In antibiotic-treated specimens, a certain portion of such cells is expected.
ACID-FAST STAIN: Stains Mycobacteria such as Mycobacterium Tuberculosis.
LIPID cell wall gives them the property of weak initial staining, plus strong retention of initial dye (like Gram+) once stained.
PROCESS:
Carbol-Fuscin dye is used initially, with heat, for 20 to 30 minutes (long time)
Acid-Alcohol is then used as a decolorizing agent.
If the organism resist the decolorization, it is considered acid-fast.
FLUORESCENT STAIN:
It has the advantage that you can scan at low power until you find something that fluoresces, then you can hone in on it.
IMMUNODIAGNOSTICS
Hemagglutination Inhibition: Certain organisms can agglutinate red blood cells. If you have antibodies to impede that agglutination, then you have a positive test result (antibody is present).
Direct Fluorescent Antibody: Fluoro tagged antibody checks for presence of antigen directly.
Indirect Fluorescent Antibody: First allow antigen-antibody reaction, then use a second Fluoro tagged antibody (anti-antibody) to identify presence of the first antibody.
Complement Fixation: Usually avoided; expensive.
ACUTE and CONVALESCENT TITERS: Take an antibody titer at initial infection (acute), and again four to five weeks later. You would expect a four-fold increase in titer if the suspected organism is indeed the infecting organism.
There is usually a baseline level of antibody present with or without infection, because of cross-reactivity with other antigens. Thus you use acute:convalescent titers as a means of comparison.
For some organisms, there are also absolute antibody levels that are diagnostic.
Radio-Immunoassay (RISA, ELISA): Take the patient's direct specimen and try to detect residual evidence of the organism's presence. Used when recovery of bacteria itself is not possible.
Molecular Probes
Polymerase Chain-Reaction (PCR): Can also be used for direct detection but it's expensive.
CULTURE:
EXCEPTIONS to the fact that organisms can be cultured in 24 hours.
Mycobacterium Tuberculosis takes up to eight weeks to culture.
Syphilis and Leprosy organisms have never been cultured.
Streaking for Isolation: Process of inoculating a plate such that you get single bacterial colonies once you culture the plate.
PROCESS: Take an inoculating stick, flame it, dip it into the bacteria (tissue sample), and then streak across the plate. Then flame it again, and streak it again.
SUSCEPTIBILITY TESTS:
KIRBY-BAUER METHOD of Antibiotic Susceptibility Testing: Testing is done using the Disk-Diffusion Method
PROCEDURE:
Grow confluent bacterial colony on plate
Add antibiotic disc in center.
Measure the Zone of Inhibition (diameter) to determine bacterial susceptibility.
RESULTS: The larger the zone of inhibition, the more susceptible are the bacteria.
Resistant: No zone of inhibition is found.
Intermediate: Intermediate zone of inhibition.
Susceptible: Larger zone of inhibition.
An interpretive table or computer, along with known concentrations of antibiotic, must be used to interpret results.
E-TEST: ANAEROBE Susceptibility Test. Strict Anaerobes must be cultures under special conditions.
Instead of a disk, the antibiotics are contained in capillary disks.
SPIRAL GRADIENT ENDPOINT TEST: Another susceptibility test
Line several isolates (can be from different patients) up on a single plate in wheel-spoke fashion.
Growth inhibition at each line can then be observed and interpreted by computer.
SERUM BACTERICIDAL TEST: Susceptibility test for patients that are extremely sick. Take serum sample and test amount of antibody actually in patient's serum. Then plate that antibody out onto culture and verify that it actually does kill the bugs at its peak and trough concentrations in the patient's serum.
A 1:8 dilution of serum inhibiting growth should be indicative that antibiotics are effective enough to kill bacteria in vivo.
MINIMUM INHIBITORY CONCENTRATION (MIC): The minimum amount of antibiotic necessary to inhibit bacterial growth to the point that no bacteria are visible with the naked eye.
This doesn't necessarily mean that no bacteria are there at all. Healthy person should be able to handle this bacteria-level with normal host-defenses.
PROCEDURE:
Take test-tubes containing varying concentrations of antibiotic, plus a control tube with no antibiotic.
Add growth media and 107 bacteria to each tube.
The most dilute tube in which no turbidity is seen is the MIC -- the minimum antibiotic concentration that inhibits bacterial growth.
MINIMUM BACTERICIDAL CONCENTRATION (MBC): Amount of antibiotic necessary to kill 99.9% of all organisms. This value must be used when treating immunocompromised patients.
PROCEDURE: To determine MBC, we must take all of our clear tubes from the MIC and culture them onto plates. Then, the plate on which the fewest number of colonies grows is termed the MBC.
Antibiotic Tolerance occurs when an isolate has a serial difference of five tubes between its MIC and MBC. Normally, the difference is only one or two tubes. This translates to a 32-fold difference in concentration.
CHOCOLATE AGAR: Heat-lysed red-blood cells in agar.
HEMOLYSIS: Appearance of culture in blood agar.
alpha-HEMOLYSIS: Partial hemolysis, appearing green.
beta-HEMOLYSIS: Full hemolysis. A "halo" appears around the colony.
gamma-HEMOLYSIS: No hemolysis.
