METHODS OF STUDYING

MICROORGANISMS
 A.DIRECT MICROSCOPE EXAMINATION

• -can often provide the etiologic diagnosis of

an infectious process and the opportunity to
initiate appropriate therapy before the result
of culture become available.
• 1.WET SMEAR
• -KOH MOUNT
• -NSS MOUNT
 B.STAINING
• Most bacteria are colorless, transparent and
difficult to see.
• Therefore, various staining methods have been
devised to enable scientist to examine bacteria.
• Specific stains and staining techniques are used
to obtain bacterial cell morphology(size, shape,
morphologic arrangement, composition of cell
wall, capsules and flagella)
 BASIC STEPS IN STAINING

• 1.FIXATION
• -the bacteria are smeared onto a glass slide,
air dried and then ”fixed”.
• -the most common methods of fixation are
heat fixation and methanol fixation.
 PURPOSE OF FIXATION

• -it kills the microorganisms

• -preserves their morphology
• -anchors the smear to the slide
 GRAM STAIN
• A gram stain is a type of microbiology or
laboratory test that determines whether
bacteria are present.
• It also determines whether bacteria
are gram negative or gram positive.
• The difference between gram negative and
gram positive bacteria can be important when
determining appropriate treatment for an
infection.
• -developed by Hans Christian Gram in 1883.
• -the color of bacteria at the end of Gram
staining procedure depends on the chemical
composition of their cell wall.
 STEPS IN GRAM STAINING
• 1.Heat-fix specimen to slide. Flood slide with
crystal violet solution(primary stain).
• 2.Rinse with water then flood with Grams
iodine(mordant).
• 3.Rinse excess iodine. Decolorize with
acetone(decolorizer).
• 4.Wash slide with water, apply safranin
(counterstain).
 RESULT INTERPRETATION
• If a bacterium is blue to purple at the end of
the procedure, it is said to be Gram positive.
• If a bacterium ends up being pink to red, it is
said to be Gram negative.
ILLUSTRATION
 ACID FAST STAINING

• -The acid-fast stain is of value in the diagnosis

of tuberculosis.
• -Acid fast bacteria are red at the end of the
acid fast staining procedure.
• -Developed by Paul Erlich in 1882.
 PROCEDURE OF ACID-FAST STAINING

• 1.Carbol fuchsin is first driven into the

bacterial cell using heat(primary stain).
• 2.Rinse with water then decolorized with acid
alcohol( decolorizer ).
• 3.Counterstain with Methylene blue
(counterstain) for 10 secs.
 INTERPRETATION
• RED COLORED BACTERIA
• -acid-fast
• BLUE COLORED BACTERIA
• -non acid fast
 SPECIMEN COLLECTION AND HANDLING

• Accurate laboratory results begin with proper

preparation on the part of the patient and
continues with proper specimen collection,
processing and handling by you, the
healthcare professional. 
• 1.Use universal precautions for collecting and
handling of all types of specimen.
• -wearing lab gowns, gloves, goggles and
face mask.
• 2.The specimen must be properly selected ,
that is it must be the appropriate type of
specimen for diagnosis of the suspected
infectious disease.
• 3.The specimen must be properly and carefully
collected.
• 4.The material should be collected from site
where the suspected pathogen is most likely
to be found.
• 5.Whenever possible, specimens should be
obtain before antimicrobial therapy has
begun.
• 6.The acute stage of the disease is the
appropriate time to collect most specimens.
• 7.Specimen collection should be performed
with care and tact to avoid harming the
patient.
• 8.A sufficient quantity of the specimen must
be obtained to provide enough material for all
required diagnostic tests.
• 9.All specimens should be placed or collected
into a sterile container to prevent
contamination of the specimen.
• 10.Specimens should be protected from heat
and cold and promptly delivered to the
laboratory.
 TYPES OF CLINICAL SPECIMENS USUALLY
SUBMITTED FOR CULTURE

• 1.BLOOD
• -consists of mixture of cells and fluid.
• -as it circulates around the body, blood is
usually sterile.
• -however, blood sometimes contain bacteria.
• BACTEREMIA
• -the presence of bacteria in the bloodstream,
may or may not be a sign of disease
• SEPTICEMIA
• -a serious disease characterized by
chills ,fever, prostration and the presence of
bacteria or their toxins in the bloodstream.
 2.CEREBROSPINAL FLUID
• CSF must be collected by means of strict
aseptic technique, both to minimize specimen
contamination and to prevent introduction of
bacteria into the CNS.
• The volume of CSF needed for culture
depends on the pathogens being sought. For
routine bacterial cultures, a few milliliters of
CSF is adequate.
3.RESPIRATORY TRACT SPECIMENS
• Expectorated sputum continues to be the
most commonly collected respiratory
specimen for bacterial cultures.
• Expectorated sputum specimens should be
screened by gram staining for contamination
with saliva.
 4.STOOL
• Optimal test results are obtained when
microbiological testing is performed on fresh
stool specimens.
• Because testing fresh specimens is impractical
in most clinical settings (particularly
outpatient settings), most stool specimens are
collected and then placed in vials containing
different transport media and fixatives.
 5.URINE
• -Urine is ordinarily sterile while it is in the
urinary bladder.
• However, during urination, it becomes
contaminated by indigenous microflora of the
distal urethra.
• -Clean- catched midstream ( CCMS) urine is
the best specimen for urine culture.
 6.WOUND SPECIMEN
• Should be an aspirate rather than a swab
specimen.
• 7.THROAT SWABS
• -Routine throat swabs are collected to
determine whether a patient has a strep
throat.
 8.GC CULTURES
• -The initials GC represents an abbreviation for
gonococci, a term referring to Neisseria
gonorrhea.
• -swabs from vagina, cervical or urethra are
submitted for culture.
• THANK YOU