BACTERIOLOGY

Bacteriology simply exists to confirm certain presence of pathogens that infests human
beings. Bacteriology quantitates the presence of organism and its certain type, whatever family it
belongs. RCH laboratory offers convenient common biochemical and culture media preparations.
They are semi-automated in their biochemical tests because they utilize BACTEC and also they
read organisms using the BBL crystal with a matching computer. They also have a biosafety
hood level II for culture and smearing operations. They also do culture and sensitivity in some
specimens to identify susceptibility to antibiotics.. This section is operated by RMTs for culture
preparations and phleb-nurses for AFB smearing.
On my first day of duty I was told to do AFB smearing with the phleb nurse, we the
smearing at around 10pm in the morning since sputum specimens are being submitted at this
time. The way we do the smearing is the same with the previous hospital I was with. A 2X3
dimension is observed with the coiling method. Sir Rodel was in charge at that moment and he
oriented me with the proper way of handling specimen and the proper PPE that should be worn.
N95 is the mask of choice and the biosafety glass is below head level. Specimens are handled as
infectious since AFB bacilli can be easily acquired through inhalation and may promote
tuberculosis. Specimens are placed on a clean glass slide using a stick with a pointed end
together with the pattern on the other side of the glass. I was also able to perform the Ziehl
neelsen method provided with the proper standard protocol of handling these infectious agents.
Ziehl neelson method or the hot method uses carbofuschin as its primary stain together with
methylene blue as its secondary stain and acid alcohol as its decolorizer. Carbofushin is handled
properly since its fumes are carcinogenic when inhaled. Furthermore, the waxy mycolic acid
containing the cell wall of MTB is relatively impermeable to ordinary staining techniques. They

can be stained by aniline dyes using drastic measures such as application of heat and phenol. The
heat applied softens the wax in the cell wall and allows the basic fuschin to enter. Once stained, it
resist decolorization by acids this is due to the fact that phenol-dye mixture us more soluble in
waxes of the mycobateria than the acid or alcohol. This way phenol acts as a mordant. The
mycobacteria retains the primary stain pink while the background material get decolorized and
takes up the methylene blue. When microscopic examination is done, a distinctive color is
observed which indicates the presence of acid fast bacilli. Furthermore, a standard grading
pattern is being followed to provide a more accurate way of reporting results (0,+n, 1+, 2+, 3+).
In gram staining preparation it uses crystal violet stain as its primary stain, which stains
everything to violet. Gram's iodine acts as a mordant that causes the crystal violet to penetrate
and adhere to the gram-positive organisms. Also, the acetone-alcohol mixture acts as the
decolorizer that washes the stain away from everything in the smear except the gram-positive
organisms. While the safranin is the counter-stain that stains everything in the smear that has
been decolorized: pus cells, mucus, gram-negative organisms. The gram-negative organisms will
stain a much deeper pink than the pus cells, and mucus will stain even lighter pink than the pus
cells. These procedure is very important in preparation for culture for further identification of the
bacteria present. I am also able to do susceptibility culture using Gram + and antimicrobials.
The activity added my experience on streaking. the same technique we use during my bacte years
in AK.

Overall what I learned in this section backs-up what I learned in school. Though for
me 1 week is not enough to learn everything in this section, but atleast the experience will
live on.

Ramiro Community Hospital

Tagbilaran City
Bohol

A Narrative Report on Bacteriology

Rouselle John E. Talingting

BSMT - INTERN