Laboratory Methods

Dr. Almurdi, DMM, MKes

 Introduction
• Bacterial diseases is diagnosed
principally by culture of the
microorganisms responsible.
 cont …
• Special techniques are necessary for
this and for subsequent identifica-
tion of the isolated organisms, often
using biochemical and serological
tests.
 Methods Used in Lab
• 1. Microscopy
• 2. Culture
• 3. Bacterial Identification
• 4. Tests of Antimicrobial Drugs
• 5. Serology
 MICROSCOPY
• Preparations
– Stained film : heat-fixed smears on slides of
specimens or bacterial cultures stained by
flooding with appropriate dyes.
– Wet film : drops of liquid specimens or fluid
cultures placed on a slide and covered with
a coverslip.
 STAINING
• Gram’s stain : the most widely used, it not
only reveals the shape and size of the bacteria
but enables classified into Gram-positive and
Gram negative
– Observe :
• Gram-positive bacteria resist decolorization and stain
blue-black
• Gram-negative bacteria are decolorized and so stain
pink with the counterstain.
 cont …
• Staining for AFB, Ziehl-Neelsen method :
concentrated carbol fuchsin (heated),
decolorize with acid and alcohol, counterstain
with methylene blue.
– Observe : for red bacilli against a blue ground
 CULTURE
• Bacteria grow well in vitro on artificial media,
the majority of pathogenic bacteria grow on
blood agar
• Growth requirements, they need organic
materials (carbohydrates, amino acids) and
sometimes specialised growth factors for
culture in lab.
 Constituents of Culture Media
• 1. Water
• 2. Sodium chloride, other electrolites
• 3. Pepton; contains proteases, amino acids
• 4. Meat extract, yeast extract : used to enrich
media; contain protein, carbohydrate,
inorganic salts, growth factor
• 5. Blood, sometimes serum
• 6. Agar, a carbohydrate derived from sea weed
 SOLID MEDIA
• Advantages and uses :
–Allows separate colony formation
–Colonial morphology (presumptively
identified)
–Quantitation
–Pure culture
 Liquid Media
• Dispensed in tubes
• Growth is recognised by turbidity in the fluid
• Fluid media with special constituents (sugars)
are widely used to test for identification.
• Enrichment media are fluids which encourage
the preferential growth of a particular
bacterium.
 Transport Medium
• For the preservation of delicate
pathogens during transit to the
laboratory.
• Stuart’s transport medium :
– A semi solid
– Non-nutrient agar with thioglycollic acid
(as reducing agents) and electrolytes
 IDENTIFICATION
• Colonial and microscopic morphology
• The condition required for growth
• Biochemical tests
• Recognition of enzymes
• Antigenic structure
• Typing of bacterial strain
 Sensitivity Tests
• Disc diffusion
– Method : paper discs impregnated with antibiotic
solution are placed on the surface of a plate
inoculated with bacterial culture under test.
• Tube dilution
– Method : a series of tube with doubling dilutions
of antibiotic in broth are inoculated with the
bacterium under test.
 SEROLOGY
• Some diseases are diagnosed by demonstra-
tion of antibody to the causal organism in the
patient’s blood.
• Titre : is the term for the highest dilution of
serum at which antibody activity is demon-
strable, usually expressed as the reciprocal of
the serum dilution.
 METHODS
• 1. Agglutination
• 2. Precipitation
• 3. Complement fixation
• 4. Immunofluorescence
• 5. ELISA
• 6. RIA
 AGGLUTINATION
• Antibody in patient’s serum is
detected by its own ability to
cause visible aggregation of
suspensions of bacteria.
 PRECIPITATION
• The antigen is in soluble form
and antibody is detected usually
by formation of a visible line of
precipitate as a result of
diffusion in agar gels.
 Complement Fixation
• Combination of antigen with antibody
fixes or uses up complement. When an
indicator system (sheep erythrocytes
coated with rabbit antibody) is then
added, haemolysis takes place if there is
free or unfixed complement.
 Immunofluorescence
• Serum antibody that has reacted
with antigen attaches the labelled
anti-human globulin, when view by
ultraviolet microscopy, the antigen
fluoresces.
 ELISA
• Like immunofluorescen, depend on
tagging antihuman globulin with an
enzyme. Antibody in patient’s serum is
detected by a color change produced by
the bound enzyme on addition of
suitable substrate.