Blood Culture

A test done to determine if there are any foreign invaders in the patient’s blood be it a bacterium, yeast,
or any microorganisms. If it turns out to be positive, that means there is foreign invader present in the
patient’s blood. If it turns out to be negative that means none is detected. Infection in the blood known
as bacteremia involves blood the circulate through out your body. Infections in the skin, liver, lungs,
urine or your gastrointestinal tract are the most common sources of bacteria. This infection can stem
out of your body and become systematic infection affecting your entire body known as sepsis. This type
of infection will interfere with the body’s normal defenses and prevent the immune system to function
properly. The pathogens will also produce harmful toxins the damages the internal organs leading to
organ failure. Sepsis is a condition caused by the response of the immune system to an infection. It
occurs when the system’s response is not in balance (affect homeostasis) triggering changes.

In situations where it turns out to be positive, the doctor will then identify what kind of invader present,
to confirm the findings, a follow up test may happen.

After confirming the identity of the invader, the doctor will use method to identify the effective
antibiotic to be used for the treatment. Bacteria may be resistant to other antibiotics as each differs in
genes. And so it is important to identify the right antibiotic for the specific bacteria for treatment. This
test is called susceptibility test/sensitivity test.

METHODS MAY INCLUDE:

1. DILUTION METHODS

The Broth dilution method involves subjecting the isolate to a series of concentrations of antimicrobial
agents in a broth environment.   Microdilution testing uses about 0.05 to 0.1 ml total broth volume and
can be conveniently performed in a microtiter format.  Macro dilution testing uses broth volumes at
about 1.0 ml in standard test tubes.  For both of these broth dilution methods, the lowest concentration
at which the isolate is completely inhibited (as evidenced by the absence of visible bacterial growth) is
recorded as the minimal inhibitory concentration or MIC.  The MIC is thus the minumum concentration
of the antibiotic that will inhibit this particular isolate.  The test is only valid if the positive control shows
growth and the negative control shows no growth. 

A procedure similar to broth dilution is agar dilution.  Agar dilution method follows the principle of
establishing the lowest concentration of the serially diluted antibiotic concentration at which bacterial
growth is still inhibited. 

2. DISK DIFFUSION METHOD

The most widely used method because of its cost, effectivity, and
efficiency. It is also known as the Kirby-Bauer test. The effectivity
of the antibiotic is determined by measuring the zone of inhibition
(the zone wherein bacterial growth is not possible). The results
depending on the measurement of the zone of inhibition will be
either susceptible (S), intermediate (I), or Resistant (R).
Resistant: 13mm or less Intermediate: 14-16 mm Susceptible: 17 mm or more
3. E-TEST

E-test (AB Biodisk, Solna, Sweden) is a commercially available test that utilizes a plastic test strip
impregnated with a gradually decreasing concentration of a particular antibiotic.  The strip also displays
a numerical scale that corresponds to the antibiotic concentration contained therein. This method
provides for a convenient quantitative test of antibiotic resistance of a clinical isolate.   However, a
separate strip is needed for each antibiotic, and therefore the cost of this method can be high. 

Gram positive vs Gram negative

Most bacteria are broadly categorized into two; Gram Positive and Gram Negative

The main component of the cell wall of bacteria is peptidoglycan. The distinct difference between gram
positive and gram negative is that gram-positive bacteria has a wall that has more peptidoglycan
compared to gram negative bacteria. This can be determined through a test called Gram Stain, this test
was discovered by Hans Christian Gram. Identifying whether a bacterium is gram positive or negative is
significant in narrowing the antibiotics to be used for treatment.

There are some bacteria that are gram-variable or gram-indeterminate. However, even this information
may be useful in narrowing down bacterial identity. The technique is most reliable when cultures are
less than 24 hours old.  The primary limitation of the technique is that it yields erroneous results if
mistakes are made in the technique.

Gram Staining Procedure

Materials

 Crystal violet (primary stain)

 Gram's iodine (mordant, to fix crystal violet in the cell wall)

 Ethanol or Acetone (decolorizer)

 Safranin (secondary stain or counterstain)

 Water in a squirt bottle or dropper bottle

 Microscope slides

 Compound microscope

Steps

1. Place a small drop of bacterial sample on a slide. Heat fix the bacteria to the slide by passing it
through the flame of a Bunsen burner three times. Applying too much heat or for too long can
melt the bacteria cell walls, distorting their shape and leading to an inaccurate result. If too little
heat is applied, the bacteria will wash off the slide during staining.

2. Use a dropper to apply the primary stain (crystal violet) to the slide and allow it to sit for 1
minute. Gently rinse the slide with water no longer than 5 seconds to remove excess stain.
 Rinsing too long can remove too much color, while not rinsing long enough may allow too much
stain to remain on gram-negative cells.

3. Use a dropper to apply Gram's iodine to the slide to fix the crystal violet to the cell wall. Let it sit
for 1 minute.

4. Rinse the slide with alcohol or acetone about 3 seconds, followed immediately with a gentle
rinse using water. The gram-negative cells will lose color, while the gram-positive cells will
remain violet or blue. However, if the decolorizer is left on too long, all cells will lose color!

5. Apply the secondary stain, safranin, and allow it to sit for 1 minute. Gently rinse with water no
longer than 5 seconds. The gram-negative cells should be stained red or pink, while the gram-
positive cells will still appear purple or blue.

6. View the slide using a compound microscope. A magnification of 500x to 1000x may be needed
to distinguish cell shape and arrangement.

Common Shapes of Bacteria

 Coccus: spherical or round

 Bacillus: rod shaped

 Spiral: curve, spiral, or twisted

Common Bacterial Cell Arrangements

 Diplo: cells remain in pairs after dividing

 Strepto: cells remain in chains after dividing

 Tetrad: cells remain in groups of four and divide in two planes

 Sarcinae: cells remain in groups of eight and divide in three planes

 Staphylo: cells remain in clusters and divide in multiple planes