HANDOUTS FOR CLINICAL MICROSCOPY AND BACTERIOLOGY

CLINICAL MICROSCOPY
Fecalysis – macrosocpic and microscopic examination of a stool sample in order to diagnose
certain conditions (esp. infections) in the gastrointestinal tract.
The ideal specimen for examination is the freshly collected specimen.
Soft specimens must be examined within 1 hour of passage.
Liquid/diarrheic specimens must be examined within 30 minutes.
Solid/formed specimens (stool) can be kept for 1 day, with overnight refrigeration if needed.
If stool examination cannot be done immediately, the specimen must be placed in fixative.
Fixatives – substances that preserve the morphology of protozoa and prevent further
development of helminth eggs and larvae.
Ratio: 3 parts fixative: 1 part stool sample
Urinalysis – physical and chemical examination of the urine in order to give clues in diagnosing
or evaluating urinary tract disorders, especially in the areas of metabolic and renal disorders
Glucose Test – based on a double sequential enzyme reaction. Glucose oxidase
catalyzes the formation of glucuronic acid and hydrogen peroxide from the oxidation of
glucose. Peroxidase catalyzes the reaction of hydrogen peroxide with a potassium iodide
chromogen to oxidize the chromogen to colors ranging from green to brown
Bilirubin Test – based on the coupling of bilirubin with diazotized dichloroaniline in a
strongly acid medium. The color ranges through various shades of tan.
Ketone Test – based on the development of colors ranging from buff-pink for negative
reading, to purple when acetoacetic acid reacts with nitroprusside.
Specific Gravity – based on the apparent pKa change of certain pretreated
polyelectrolytes in relation to ionic concentration. In the presence of an indicator, color
range from deep blue-green in the urine of low ionic concentration through green and
yellow-green in urine of increasing ionic concentration.
Blood – based on the peroxidase-like activity of hemoglobin, which catalyzes the
reaction to diisopropylbenzene and 3,3’, 5,5’-tetramethylbenzidine. The resulting color
ranges from orange through green; very high levels of blood may cause the color
development to continue to blue.
RBC Morphology is performed if the RBCs detected are more than 4 per HPF.
 • Isomorphic RBCs – morphological features are uniform and no more than
two types; these morphologies result from renal pelvis, ureter, or bladder
bleeding (non-glomerular bleeding)
• Dysmorphic RBCs – variously shaped blebs or projections at the cell
membrane; these morphologies result from glomerular bleeding

pH – based on a double indicator principle that gives a broad range of colors covering
the entire urinary pH range.
Protein – based on the protein-error-of-indicators principle. At a constant pH, the
development of any green color is due to the presence of protein.
Nitrite - depends upon the conversion of nitrate (derived from the diet) to nitrite by the
action of gram-negative bacteria in the urine. At the acid pH of the reagent area, nitrite
in the urine reacts with p-arsanilic acid to form a diazonium compound. This diazonium
compound in turn couples with 1,2,3,4-tetrahydrobenzo(h)quinolin-3-ol to produce pink
color
Leukocytes – contain esterase that catalyze the hydrolysis of the derivatized pyrrole
amino acid ester to liberate 3-hydrozy-5-phenyl pyrrole. The pyrrole then reacts with a
diazonium salt to produce a purple product

BACTERIOLOGY
Gram Staining – ability of bacterial cell wall to retain the primary stain (Crystal Violet) during
solvent treatment. Gram-positive microorganisms are able to retain the crystal violet due to
their cell walls having higher peptidoglycan component. Gram-negative microorganisms, on the
other hand, have lower peptidoglycan component and higher lipid component in their cell
walls.
Note: The length of decolorization is a critical step in gram staining as prolonged exposure to a
decolorizing agent can remove all the stains from both types of bacteria.
Culture and Sensitivity – done in order to identify microorganisms by means of their growth in
specific culture media and/or enrichment broths, and in order to assess the sensitivity or
resistance of certain bacteria/microorganisms in specific antibiotics.
This is usually done by culturing the collected specimens in plated cultures/enrichment broths
and see if there will be any growth in that specific medium/broth.
Checking the sensitivity to specific antibiotics is traditionally done via Kirby-Bauer testing. In
this method, bacteria are cultured in growth medium and antibiotic discs are added to the
plate. Areas of clear media surrounding the disc indicate inhibition of bacterial growth after
allowing overnight growth of the bacteria.

Kirby-Bauer Testing
 CULTURE MEDIA/ENRICHMENT BROTHS USED
SPECIMEN MEDIA ENRICHMENT BROTH ATMOSPHERE REQ’T
BLOOD CAP, BAP, MAC N/A 5% CO2
INCUBATOR (CAP, BAP)
AMBIENT (MAC)
URINE BAP, MAC AMBIENT INCUBATOR
CSF CAP, BAP, MAC BHI
SPUTUM CAP, BAP, MAC, GBA, BCA 5% CO2
INCUBATOR (CAP, BAP)
ETA CAP, BAP, MAC, GBA, BCA AMBIENT (MAC)
BRONCHOALVEOLAR CAP, BAP, MAC
LAVAGE
THROAT SWAB BAP 5% CO2 INCUBATOR
EFFUSION (PLEURAL,
PERITONEAL, SYNOVIAL, CAP, BAP, MAC BHI
5% CO2
PERICARDIAL, HYDROCOELE)
INCUBATOR (CAP, BAP)
URETHRAL, VAGINAL, CAP, BAP, MAC
AMBIENT (MAC)
PENILE, ANORECTAL MTM (PENILE DISCHARGE) THIO
DISCHARGE
TISSUE CAP, BAP, MAC THIO
WOUND DISCHARGE (SWAB) BAP, MAC THIO
WOUND DISCHARGE BAP, MAC THIO
AMBIENT INCUBATOR
ABSCESS; EXUDATES
STOOL BAP, MAC, TCBS, SSA APW, SEL-F

India Ink Staining – mostly used for Cryptococcus sp. detection. Since India ink is an acidic dye,
it repels on the cell wall of bacteria, staining the glass slide black but bacteria cells will remain
clear.
Toxoplasma, Monilia, Gram Staining (TMG) – microscopic evaluation and detection of
microorganisms such as Toxoplasma sp., Candida (Monilia) sp., and gram-positive/gram-
negative organisms in cervical swabs/vaginal discharge by Gram-staining.