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Bacteriology Review

Bacteriology Review

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Published by: yachiru121 on May 22, 2012
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 www.brain101.info
1
BACTERIOLOGYLABORATORY REPORTS / TECHNIQUESASEPTIC TECHNIQUE:
In handling bacteria specimens, do everything possible to assure that extraneous bacteria
 from you
do notcontaminate 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:
o
 
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.
o
 
CHELATING AGENT: Add
Gram's Iodine
which will adhere only to cell walls that have a lot of peptidoglycan inthem (i.e. Gram+)
o
 
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.
o
 
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
.
o
 
FALSE GRAM-NEGATIVES:
 
 Dead Gram+ cells will appear as though they are Gram-
. In antibiotic-treatedspecimens, a certain portion of such cells is expected.
 
ACID-FAST STAIN:
Stains Mycobacteria such as
 Mycobacterium Tuberculosis
.
o
 
LIPID cell wall gives them the property of 
weak initial staining 
, plus
 strong retention of initial dye
(like Gram+)once stained.
o
 
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:
o
 
It has the advantage that you can scan at low power until you find something that fluoresces, then you can hone inon it.
 
IMMUNODIAGNOSTICS
o
 
Hemagglutination Inhibition:
Certain organisms can agglutinate red blood cells. If you have antibodies to impedethat agglutination, then you have a positive test result (antibody is present).
o
 
Direct Fluorescent Antibody:
Fluoro tagged antibody checks for presence of antigen directly.
o
 
Indirect Fluorescent Antibody:
First allow antigen-antibody reaction, then use a second Fluoro tagged antibody(anti-antibody) to identify presence of the first antibody.
o
 
Complement Fixation:
Usually avoided; expensive.
o
 
ACUTE and CONVALESCENT TITERS:
Take an antibody titer at initial infection (acute), and again four to fiveweeks later. You would expect a
 four-fold increase in titer 
if the suspected organism is indeed the infectingorganism.
 
There is usually a baseline level of antibody present with or without infection, because of cross-reactivitywith 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.
o
 
Radio-Immunoassay (RISA, ELISA):
Take the patient's direct specimen and try to detect residual evidence of theorganism'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.
o
 
 Mycobacterium Tuberculosis
takes up to eight weeks to culture.
o
 
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.
 
 www.brain101.info
2
o
 
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:
o
 
KIRBY-BAUER METHOD of Antibiotic Susceptibility Testing
: Testing is done using the
Disk-DiffusionMethod
 
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 tointerpret results.
o
 
E-TEST
: ANAEROBE Susceptibility Test. Strict Anaerobes must be cultures under special conditions.
 
Instead of a disk, the antibiotics are contained in capillary disks.
o
 
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.
o
 
SERUM BACTERICIDAL TEST:
Susceptibility test for patients that are extremely sick. Take serum sample andtest amount of antibody actually in patient's serum. Then plate that antibody out onto culture and verify that itactually 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 bacterialgrowth to the point that
no bacteria are visible with the naked eye
.
o
 
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.
o
 
PROCEDURE:
 
Take test-tubes containing varying concentrations of antibiotic, plus a control tube with no antibiotic.
 
Add growth media and 10
7
bacteria to each tube.
 
The most dilute tube in which no turbidity is seen is the MIC -- the minimum antibiotic concentration thatinhibits bacterial growth.
o
 
MINIMUM BACTERICIDAL CONCENTRATION (MBC):
Amount of antibiotic necessary to kill 99.9% of allorganisms. 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 themonto plates. Then, the plate on which the fewest number of colonies grows is termed the MBC.
o
 
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.
 
 www.brain101.info
3
EPIDEMIOLOGY / GENERAL STUFF
Causes of COMMUNITY-ACQUIRED PNEUMONIA:1.
 
Streptococcus Pneumoniae
Causes of NEONATAL (less than 3 months old) MENINGITIS and SEPSIS: In order 1.
 
Group-B
Strep
2.
 
 Escherichia Coli
Causes of INFANTILE (infants 6 - 24 months) MENINGITIS and SEPSIS: In order 1.
 
 Haemophilus Influenzae
(Hib)2.
 
 Neisseria Meningitidis
Causes of UTI's: In order 1.
 
 E. Coli
2.
 
 Proteus Vulgaris / Mirabilis
:
o
 
10-15% of hospital acquired UTI's.3.
 
 Pseudomonas Aeruginosa
ENTEROBACTERIACEAE: Cause Gastroenteritis, UTI's, Pneumonia, and Bacteremia.
 
GASTROENTERITIS: STRICTLY LUMINAL1.
 
Enteropathogenic
 E. Coli
(EPEC)2.
 
Enterotoxigenic
 E. Coli
(ETEC)3.
 
Vibrio Cholerae
4.
 
Vibrio Parahaemolyticus
(mechanism uncertain)
 
GASTROENTERITIS: INVASIVE (DYSENTERY)1.
 
Enteroinvasive
 E. Coli
(EIEC)2.
 
Shigellosis3.
 
Salmonella Enterica
4.
 
Campylobacter Jejuni
 
BACTEREMIA1.
 
Entero hemolytic
 E. Coli
(EHEC)2.
 
Salmonella Typhi
3.
 
Salmonella Choleraesuis
 
URINARY TRACT INFECTION1.
 
 E. Coli
2.
 
 Proteus
3.
 
 Pseudomonas
4.
 
 Klebsiella
 
PNEUMONIA1.
 
 Pseudomonas Aeruginosa
2.
 
 Klebsiella Pneumoniae
ENTEROBACTERIACEAE ANTIGENIC MARKERS:
 
O-Antigen
: Carbohydrate in the cell wall.
 
K-Antigen
: Capsular antigen, when present.
 
H-Antigen
: Flagellar antigen, when present:
o
 
Campylobacter, Helicobacter, Escherichia.
 
F-Antigen
: Pili antigen.

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